Antimicrobials 1/2

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  • Created by: LBCW0502
  • Created on: 17-02-20 19:00
What are the groups of microbes?
Viruses (not cellular), fungi/microscopic eukaryotes (cellular), bacteria (cellular). Require microscopy to view individual microbes (cannot be seen with the naked eye. Each class of microbe has members that can cause human infection
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What are the two classes of microscopy?
LM and EM (Uses a beam of electrons to create an image. It is capable of much higher magnifications and has a greater resolving power than a light microscope)
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Describe features of viruses (1)
Grown in cells, infectious agents, many different viral pathogens have small genomes (few potential antiviral targets). E.g. rhinovirus, influenza, herpes virus, ebola virus
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Describe features of viruses (2)
Potential for rapid spread. Potential for high fatality rate (e.g. new influenza virus from Mexico) and often a lack of treatment options (e.g. nipah virus). Significant problem if traits combine - localised severe infections, worldwide, spread
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Describe features of fungi and other microscopic eukaryotes (1)
Have nuclear membrane, cell 'machinery' is like human cells, harder to treat by antimicrobial agents. Many form resting stages or spores (more difficult to deal with)
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Describe features of fungi and other microscopic eukaryotes (2)
Fungi - grow as single cells (yeast) or as long branches (hyphae). Some fungi are dimorphic (grow as yeasts or hyphae). Most clinical concern - Candida albicans, Aspergillus fumigatus. Invasive infections/immunocompromised
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Describe features of fungi and other microscopic eukaryotes (3)
Number of fungal infections increasing in the UK
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Describe features of fungi and other microscopic eukaryotes - parasites
Primarily protozoa e.g. Plasmodium falciparum which causes the most dangerous form of malaria. Complex life cycle (vector/mosquito). Solutions (antimicrobial/antivector). Issue with malaria in the UK (climate change)
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Describe features of bacteria (1)
Biggest clinical problem. Bacterial cells (and archaeal cells) are prokaryotes, lack nuclear membrane, grow as single cells outside human cells. Over 35,000 species each with over 1000 genes per genome
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Describe features of bacteria (2)
Divided into Gram positive and Gram negative cell types (“Gram positives” or “Gram negatives”). Reflects different cell wall structures, detected by chemical staining
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Describe features of bacteria (3)
Gram positive cell wall - thick peptidoglycan e.g. Staphylococcus aureus. Gram negative cell wall - extra liposaccharide membrane e.g. Escherichia coli
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Outline the taxonomy and nomenclature (1)
All living organisms (i.e. not viruses) classified into a structure from Domain level to species level and have two names - the first a genus name, the second a species name e.g humans - Homo sapiens
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Outline the taxonomy and nomenclature (2)
This includes bacterial species, named on their phenotype, isolation source or honouring someone
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Outline the taxonomy and nomenclature (3)
Humans (Eukarya - Animalia/Metazoa - Chordata - Mammalia - Primates - Hominidae - Homo - Homo sapiens)
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Outline the taxonomy and nomenclature (4)
Staphylococcus aureus (Bacteria - Firmicutes - Bacilli - Bacillales - Staphylococcaceae - Staphylococcus - Staphylococcus aureus)
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Outline the taxonomy and nomenclature (5)
Escherichia coli (Bacteria - Proteobacteria - Gamma-proteobacteria - Enterobacteriales - Enterobacteriaceae - Escherichia - Escherichia coli)
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Describe features of bacterial resistance (1)
nfections by certain species such as Staphylococcus aureus in pneumonia cause clinical concern because they can lead rapidly to mortality – days
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Describe features of bacterial resistance (2)
Resistance to antimicrobial agents makes certain species difficult to treat – these include: MRSA, Pseudomonas aeruginosa, Mycobacterium tuberculosis, Clostridium difficile, Gram -ve cells that have extended spectrum beta lactamase (ESBL) resistance
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Describe features of bacterial resistance (3)
Some bacterial species produce spores – resting stages for bacterial cells. “normal” cells grow out of spores – this is desporulation. Spores can remain dormant for years and are hard to destroy e.g. C.difficile, Bacillus anthracis - bioweapon
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How many microbes are there in the natural environment?
10^30, reservoir of genetic diversity, potential for spread of infection
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How many bacterial cells are in the human body?
10^14 cells. Main locations - oral cavity, upper respiratory tract, skin, GI tract
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What are the reasons for high abundance of bacteria? (1)
Bacterial species can grow rapidly e.g. cell division can be every 20 minutes, large populations can be achieved quickly. Different species have diverse physiologies, extremes of growth possible from -5-20 degrees Celsius, pH 1-11, anaerobic/aerobic
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What are the reasons for high abundance of bacteria? (2)
Bacteria can exploit many different habitats through these diverse physiologies. These diverse physiologies result from diverse functions carried in bacterial genomes
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What are the reasons for high abundance of bacteria? (3)
Microbes are diverse and widespread. Unless we do something about it, all surfaces (biotic or abiotic) and substances have microbes – this can lead to infection, to product spoilage etc. Therefore, we need to develop antimicrobial strategies
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How does an antimicrobial strategy vary by context? (1)
Pharmaceutical preparations (If microbes are present, an obvious potential to cause infection, more subtly, microbial modification/ spoilage of the drug itself?) - sterile
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How does an antimicrobial strategy vary by context? (2)
Managing microbes (including pathogens in environments e.g. care facilities/ clinics/ pharmacies/ hospitals etc, impractical to be free of microbes) - disinfectant/antiseptic
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How does an antimicrobial strategy vary by context? (3)
Infections in/on the body (there are “beneficial” bacterial cells in/ on the body as well as the pathogen we’re trying to treat, strategies needed to deal with pathogen – and ideally only the pathogen) - antibiotics
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Describe the sterilisation of microbes (1)
The complete removal of microbes or damaging microbes such that all are killed. Absolute. Sterilisation of microbes does not use antimicrobial agents. Of critical importance in relation to drug preparation
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Describe the sterilisation of microbes (2)
Methods - heat (dry/wet), ionisation radiation (gamma-radiation from 60 Co source), filter sterilisation. These strategies normally “indiscriminately” damage microbial cells/viruses (can't use strategies in humans)
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Describe features of normal bacterial growth in the laboratory (1)
Inoculate a sterile clear growth medium with a few cells. Over time, bacteria grow, turn medium cloudy. High cell numbers- 10^10/ml. If you count cells over time, normal graph emerges as 4 distinct phases – typical for “all' species (plots)
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Describe features of normal bacterial growth in the laboratory (2)
Link between normal growth, bacteriostasis and bactericide
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What are bacteriostatic/fungistatic agents?
Agents that stop bacterial or fungal growth are called bacteriostatic or fungistatic
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What are bactericidal/fungicidal/virucidal agents?
Agents that kill bacteria, fungi or viruses are called bactericidal, fungicidal or virucidal
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Describe features of disinfection (1)
Disinfection reduces the number of viable microorganisms to a level specified as appropriate. Normally focus is to reduce the number of potential pathogens to the point where they don’t represent a hazard
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Describe features of disinfection (2)
Less “clear cut” then than sterilisation. Sterilisation > disinfection.
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Describe features of disinfection (3)
Typically, these compounds are used on inanimate objects as they are still too toxic or corrosive though for use in or on humans. Concentration dependent
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Describe features of antisepsis (1)
Antiseptics (considered for use on humans). Destruction/inhibition of microbes in/on living tissue. Some disinfectants are used as antiseptics through concentrations that do not injure the host
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Describe features of antisepsis (2)
Antimicrobial agents act to prevent sepsis (growth of harmful microbes on living tissue). Sterilisation > disinfection > antisepsis
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Describe features of antisepsis (3)
For practical purposes, antiseptics can routinely be thought of as topical agents – for use primarily on skin and mucous membranes
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Describe features of preservation (1)
The prevention of the multiplication of microbes in products. We’ve accepted that microbes will be in products, but we want to limit their growth. Sterilisation > disinfection> antisepsis> preservation
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Describe features of preservation (2)
Preservatives are used in products that are either injected, ingested or applied topically to humans (pharmaceuticals, food, cosmetics, soaps etc)
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What are the practicalities? (1)
Little profit – therefore little inclination – to find novel disinfectants, antiseptics and preservatives. Often little is known about the mechanism of action of many antimicrobial agents
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What are the practicalities? (2)
Working out the mechanisms of action that disinfectants, antiseptics and preservatives have is difficult to research
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What can be used as antimicrobial agents? (1)
Aldehydes. Oxygen species. Halogens. Phenolic compounds. Alcohols. Biguanide-based compounds. Quaternary ammonium-based compounds. Organic acids
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What can be used as antimicrobial agents? (2)
Aldehydes most aggressive and organic acids least aggressive (depends on concentration). Aldehydes – exemplifying disinfectants. Biguanides – antiseptics. Organic acids – preservatives
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State features of aldehydes
The three aldehyde–based compounds used mainly are - formaldehyde, glutaraldehyde, OPA, glutaraldehyde (similar in use)
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State features of glutaraldehyde (1)
Liquid disinfectant. Treats surfaces and temperature-sensitive medical/dental equipment. MOA - cross links proteins (presents synthesis of microbial macromolecules). Glutaraldehyde binds amino groups in microbial proteins >> forms crosslinks
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State features of glutaraldehyde (2)
These crosslinks stop functions of cellular and viral proteins. Glutaraldehyde shows a broad spectrum of efficacy. Rapidly bactericidal, virucidal and fungicidal. Also has sporocidal activity
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State features of glutaraldehyde (3)
This cidal activity is typically minutes for cells/viruses, but hours for spores. Glutaraldehyde is so active it can be used to sterilise i.e. is a chemosterilant
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State features of formaldehyde (1)
Even more active than glutaraldehyde. Mode of action is similar to glutaraldehyde. Additionally though, it complexes with nucleic acids. Rapidly stops microbial functions
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State features of formaldehyde (2)
Formaldehyde is bactericidal, virucidal, fungicidal and sporocidal
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Describe features of biguanide based compounds (1)
E.g. chlorhexidine. Antiseptic uses in dentistry (e.g. mouthwash) and medicine (wound treatment). Chlorhexidine inserts directly into either bacterial or yeast cell membranes - leads to cytoplasm content leakage, loss of cell structure/function
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Describe features of biguanide based compounds (2)
Broad spectrum antibacterial activity though less effective against fungi. Not sporocidal, but inhibits desporulation and outgrowth. Limited activity against only certain viruses (compare to formaldehydes)
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Describe features of organic acids (1)
Acids are substances that dissolve in water to provide hydrogen (H+) ions. Strong acids e.g. HCl are effective antimicrobials but are limited by safety concerns and as they damage the material being treated.
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Describe features of organic acids (2)
E.g. benzoic acid, acetic acid, methylparaben (used in food and cosmetic preparations). MOA unclear - may disrupt the uptake/ transport of nutrients by microbes
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Describe features of organic acids (3)
Activity Methylparaben like other parabens are fungistatic and bacteriostatic for Gram positive cells though less effective for Gram negative cells. Other acids are very varied in their efficacies with a tendency to be microbistatic (biguanides)
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State features of Paul Ehrlich (1)
Working on the ways that analine dyes stained tissues. In the 1870s, when Koch was the first person to culture microbes on solid growth media.
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State features of Paul Ehrlich (2)
Ehrlich found that some dyes bound to certain cell types but not others. Parasites vs. human cells. Rather than just staining microbes, Ehrlich wondered if these dyes could act as “magic bullets” to kill microbes
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State features of Paul Ehrlich (3)
Could they also have selective toxicity – to be more toxic to microbial cells than human cells. This is an important concept. Harder to find targets for fungal & parasitic infections (eukaryotic cells) than bacterial infections.
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What is antibiosis?
The association of two organisms in which at least one is harmed or killed by the other. Not a new concept - first defined in 1889 by Paul Vuillemin. However, the irony is that the first “antibiotic” as we use the word was found by accident.
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How was the first antibiotic discovered? (1)
In 1928, Fleming had been studying variants of Staphylococcus aureus. After a holiday, he saw a fungal contaminant (a Penicillium sp) was present on one plate
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How was the first antibiotic discovered? (2)
What raised this to be an important moment was that he understood the significance of the extensive lysis of the bacterial colonies close to the fungus and that the colonies further away were not affected
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How was the first antibiotic discovered? (3)
Worth stressing – no “plan” to derive an antibiotic. Good fortune. A lot of work was needed though to turn that observation into a product
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How was the first antibiotic discovered? (4)
Though other antibiotics had been devised, the first humans were treated in a systematic manner with penicillin in 1941
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Describe features of antibiotic discovery (1)
Though less than eighty years ago, there are serious concerns about our ability to use antibiotics effectively in the future - running out of antibiotics. Penicillin, the first antibiotic, was discovered in 1920s
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Describe features of antibiotic discovery (2)
Rapid appreciation of antibiotics treating some infections. Antibiotic resistance was recognised early on - didn’t block new antibiotic discovery projects
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Describe features of antibiotic discovery (3)
Immediate interest in novel antibiotics (Selman Waksman). As such, from the 1940s, there was a steady increase in the numbers and sorts of antibiotic developed - aminoglycosides, teracyclines, chloramphenicol, macrolides, glycopeptides, rifamycin
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Describe features of antibiotics (1)
Natural substances. Produced typically as secondary metabolites by certain microbes inc. Streptomyces sp, Bacillus sp, Penicillium sp, Cephalosporium sp
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Describe features of antibiotics (2)
Secondary metabolites – compounds produced after the active phase of growth – so produced in stationary phase (bacterial growth curve)
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Describe features of antibiotics (3)
Secondary metabolites - often biologically-active small molecules, not required for viability, but may provide a competitive advantage to a producing organism
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Describe features of antibiotics (4)
In the 1940s, the term “antibiotic” defined a substance produced by one microorganism, which in low concentrations, inhibited the growth of other microorganisms. Only naturally- occurring substances could be classed as antibiotics
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Describe features of antibiotics (5)
Once an antibiotic has been discovered however, its chemical structure can be worked out - from this, new variants can be made by changing different sections of the antibiotic structure
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How are hybrids formed?
By chemically altering the original antibiotic molecule are called semisynthetic antibiotics (not derived from cultured species)
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Describe features of pathogen identification (1)
Patient - sample - clinical microbiologist (lab techniques, sampling strategy, preliminary results). Infectious-diseases physician (patient examination, sampling strategy, preliminary results). Diagnosis, treatment.
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Describe features of pathogen identification (2)
Patient examination – first key step – narrows the disease/ pathogen options and so helps diagnosis. This informs the Diagnostic microbiology laboratory.
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Describe features of diagnostic microbiology laboratory (1)
When a sample gets received. The suspected infection/ specimen type + microscopy informs what pathogens considered likely in the sample. This indicates what - growth conditions should be used, growth media should be used
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Describe features of diagnostic microbiology laboratory (2)
After incubation, colonies of the pathogen are recognised by their phenotype – this tentatively identifies the pathogen
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Describe features of diagnostic microbiology laboratory (3)
Microscopy helps support or rule out this identity based on cell shapes and staining.
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Describe features of bacterial cell shapes (1)
Two main forms - bacillus/coccus. Bacterial cell division results in chains or bunches of cells (Strep/ Staph). Other forms (Vibrio or spirillum) less common, but useful diagnostically. Gram staining increases the likelihood of the identification.
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Describe features of bacterial cell shapes (2)
Based on differential dye retention, bacterial species are divided into Gram positive (original dye retained) and Gram negative species. (Bacterial cell walls - two types +/-)
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How is the primary isolation phase obtained?
Bacterial culture combined with microscopy often shows a mix of species – primary isolation phase. Skill needed to identify the pathogen in this mix. The next step is to derive a pure culture of it. List pathogens (based on +/-, cocci, rods)
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What are the four strategies of action for antibiotics? (1)
Disrupting bacterial cell walls (work on peptidoglycan) e.g. beta lactams - penicllins, cephalosporins, carbapenems
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What are the four strategies of action for antibiotics? (2)
Inhibiting protein synthesis (work on the ribosome) e.g. aminoglycosides, tetracyclines, chloramphenicol, macrolides, clindamycin
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What are the four strategies of action for antibiotics? (3)
Inhibiting nucleic acid synthesis (coiling, RNAP, cytotoxic) e.g. quinolones, rifampicin, metronidazole
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What are the four strategies of action for antibiotics? (4)
Antimetabolite activity (dihydrofolate reductase.activity) e.g. trimethoprim
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What do we want to achieve with the use of antibiotics?
Drug treatment used, bacteria die, person is healthy again (don't want bacteria to continue to spread/person remains ill, resistance)
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What are the two forms of antibiotic resistance? (1)
Intrinsic – resistance occurs “naturally”. Microbes that for example lack the specific target for a particular antibiotic. Tends to hold for all isolates of a species
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What are the two forms of antibiotic resistance? (2)
Acquired - mutational resistance. Horizontal transfer – when a means of resisting an antibiotic in one species passes to another previously susceptible species - mobile genetic elements
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What are the two forms of antibiotic resistance? (3)
Acquired antibiotic resistance esp horizontal transfer is a great concern
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What is the impact of gene transfer on antibiotic susceptibility?
Resistant/non-resistant bacteria exist, bacterium multiply by billions, non-resistant bacteria receive new DNA, drug resistant bacteria multiply/thrive
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Describe features of antibiotic spectrum (1)
Hard to define, refers to the range of species potentially susceptible. Often described as narrow or broad depending on how wide this range is
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Describe features of antibiotic spectrum (2)
Broad spectrum are valuable when no knowledge of the identity of the pathogen e.g. when no time to find this out
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Describe features of antibiotic spectrum (3)
When we come to talk about antibiotics acting on species – “good” or “active” means likely to be effective. Tetracyclines - broad spectrum (more complex, effectiveness varies, species less susceptible)
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Describe features of antibiotic spectrum (4)
Antibiotic resistance of UTIs, E.coli. A further complication is that even at one time point, not all strains of a species behave in the same way, MRSA
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Describe features of reserved antibiotics
These are those remaining effective for difficult to treat species and are kept for these infections. For example, “glycopeptide antibiotics reserved as a last line of defence against resistant Gram-positive infections”
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Which terms come together when considering which antibiotics could be used to treat an infection?
Antibiotic resistance and antibiotic spectrum
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How is an antibiotic chosen? (1)
Clinical sample >> pathogen. Identified to species level as a pure culture as soon as possible. This narrows down what antibiotics might be potentially effective
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How is an antibiotic chosen? (2)
Then for each particular strain, you need to make a specific assessment e.g. by Kirby Bauer disc diffusion assays here as to what agent(s) to recommend. Other clinical factors are considered and treatment begun
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How is an antibiotic chosen? (3)
Antibiotics are broadly regarded either as - bacteriostatic (bacterial growth is inhibited) or bactericidal (bacterial cells are killed)
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How is an antibiotic chosen? (4)
Lowest antibiotic concentration that inhibits the growth of the isolate being tested is the Minimum Inhibitory Concentration (MIC)
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How is an antibiotic chosen? (5)
If relevant, the lowest concentration that kills 99.9% of the cells is the Minimum Bactericidal Concentration (MBC)
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What does a bacterial cell need in order to grow? (1)
Resources (nutrients), make proteins (enzymes to make cellular components), needs to divide (bacteria divide by binary fission, where replication of the chromosome triggers cell division into two daughter cells), need to be intact
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What does a bacterial cell need in order to grow? (2)
Interfering with any these needs has potential for “success”
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Reinterpret the requirements for bacterial cell growth in regards an an effective antibiotic in a human (1)
Needs water and other resources (are there specific resources that bacteria need but humans don’t?). Needs to make proteins e.g. enzymes able to make cellular components (can bacterial protein production be stopped or adversely affected?)
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Reinterpret the requirements for bacterial cell growth in regards an an effective antibiotic in a human (2)
Needs to divide – bacteria divide by binary fission, where replication of the chromosome triggers cell division into two daughter cells (can the bacterial chromosome be adversely affected?)
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Reinterpret the requirements for bacterial cell growth in regards an an effective antibiotic in a human (3)
Needs to be intact (can we break open bacterial cells but not damage our own cells?) - selective toxicity
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State components in a bacterial cell
More simple than eukaryotes cells. Unicellular organisms (no “protection” from external stresses). Ribosomes for protein synthesis. Cytoplasm, ribosomes, plasmid, cell wall, cell membrane, chromosomes, flagella
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Most antibiotics use which two strategies? (1)
Disrupt bacterial cell walls. Inhibit protein synthesis. (Inhibit nucleic acid synthesis, antimetabolite). Each of these routes is fundamental to the bacterial cell and in turn the next generation of bacterial cells
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Most antibiotics use which two strategies? (2)
Start with the bacterial cell wall and its structural component peptidoglycan in relation to antibiotics
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Why target bacterial cell walls? (1)
A defective or no cell wall compromises both growth and ability of bacteria to divide. Implies less fit cells or dead cells
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Why target bacterial cell walls? (2)
Bacterial cell walls are constructed with peptidoglycan in similar ways. This polymer gives bacterial cell walls structural rigidity yet flexibility. With no cell wall, bacteria are very osmotically fragile
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Why target bacterial cell walls? (3)
The Gram stain is the traditional starting point for bacterial identification. Used to classify cells as either Gram-positive or Gram-negative based on differential dye retention - recognise structural difference
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Why target bacterial cell walls? (4)
Gram positive cells have no outer membrane and more (c. 30nm) of peptidoglycan than Gram negative cells (c. 3nm)
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Why target bacterial cell walls? (5)
The wall itself is made of peptidoglycan (also murein or muropeptide)
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Describe features of bacterial cell walls (1)
Established cell walls containing peptidoglycan. When dividing for growth, bacteria need a new cell wall. Means synthesising fresh peptidoglycan. Peptidoglycan is unique to bacteria and is not in eukaryotic cells. A good target – selective toxicity
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Describe features of bacterial cell walls (2)
Structurally, peptidoglycan is a chain of alternating molecules of N- acetylglucosamine and N-acetylmuramic acid of 10-65 disaccharide residues
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Describe features of bacterial cell walls (3)
On the N-acetylmuramic acid there is a short peptide chain (3 to 5 aa). in E. coli this is often L-Ala-D-Glu-L-Lys*-D-Ala-D-Ala
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Describe features of bacterial cell walls (4)
Differs from species to species, though D-Ala D-Ala very common. The peptide bridges formed by transpeptidases between the strands of the polymers create a rigid, yet flexible coating for the cell.
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Describe features of bacterial cell wall synthesis (1)
Linear strands are cross-linked by transpeptidation. Peptidoglycan monomers attaches to growing linear strand. Peptidoglycan subunits cross the membrane attached to bactoprenol a large lipid carrier (joined by a pyrophosphate bridge).
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Describe features of bacterial cell wall synthesis (2)
Peptidoglycan (here muropeptide) subunits are assembled in the cytoplasm.
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Give examples of two types of antibiotics that disrupt cell walls
Beta lactams, glycopeptides (other antibiotics such as polypeptides (e.g. bacitracin) target cell walls). Even for beta lactams and glycopeptides, there is a huge number of individual antibiotics
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Describe features of beta lactam antibiotics (1)
The chains of peptidoglycan are built and cross linked by a range of bacterial enzymes including transpeptidases and transglycosylases. Penicillin-binding proteins include transpeptidases that can be bound by Beta lactam antibiotics
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Describe features of beta lactam antibiotics (2)
When these antibiotics are given to a growing cell, the beta lactams bind to these proteins. This blocks the formation of cross-links between the peptidoglycan chains - in turn leads to increased internal osmotic pressure, activates autolysis
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Describe features of beta lactam antibiotics (3)
β-lactams inhibit transpeptidation by binding to PBPs on maturing peptidoglycan strands. Penetration of beta lactams into the cell wall, leads to the death of the bacterial cell, beta lactams are bactericidal
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What are the three sub groups within beta lactams?
Penicillins, cephalosporins, carbapenems
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State features of penicillins
Four different structures of penicillins were originally isolated – these were labelled F, G, K and X. Of these, penicillin G seemed to have the best properties and was easiest to derive. Penicillin G is now commonly known as benzylpenicillin (SAR)
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