4.3 - Antimicrobial Therapies

Question 1

What is an antibiotic?

Answer 1

• an antibiotic is an antimicrobial agent produced by a microorganism that kills or inhibits other microorganisms
• most antibiotics in use today are produced by soil-dwelling fungi or bacteria
• antibiotics synthesised today encompass a range of natural, semi-synthetic and synthetic chemicals with antimicrobial activity
• antimicrobial - chemical that selectively kills or inhibits microbes
• bactericidal - kills bacteria
• bacteriostatic - stops bacteria growing
• antiseptic - chemical that kills or inhibits microbes that is usually used typically to prevent infection

Question 2

What is resistance?

Answer 2

• minimal inhibitory concentration (MIC) - the lowest concentration of antibiotic required to inhibit growth
• breakpoint - clinically achievable concentration - defines whether bacteria is susceptible or resistant
• if MIC </= breakpoint, then the bacteria is considered susceptible to that antibiotic
• antibiotic resistance evolved through natural selection
• population contains cells with AB resistance due to mutations/acquired DNA
• in absence of selection pressure (e.g. ABs), AB resistant chains have no advantage = low prevalence of AB resistant chains in patient population
• in presence of selection pressure (e.g. ABs), it is advantageous = high prevalence of AB resistant strains in patient population
• natural selection has been driving antibiotic production and the development of resistance mechanisms for millions of years
• human use has provided strong selective pressure for the acquisition or development of AB resistant genes

Question 3

Misconceptions at the dawn of the antibiotic era

Answer 3

• resistance against more than one class of antibiotics at the same time would not occur
• horizontal gene transfer would not occur
• resistant organisms would be significantly less ‘fit’

Question 4

What does AB resistance lead to?

Answer 4

• increased mortality, morbidity and cost
• increased time to effective therapy
• requirement for additional approaches e.g. surgery
• use of expensive therapy e.g. drugs
• use of toxic drugs e.g. vancomycin
• use of less effective ‘second choice’ antibiotics

Question 5

Major AB resistant bacterial pathogens - gram negative

Answer 5

• Pseudomonas aeruginosa - cystic fibrosis, burn wound infections - survives on antibiotic surfaces
• E. Coli (ESBL), Klebsiella spp. - GI infection, neonatal meningitis, septicaemia, UTI
• Salmonella spp. - GI infection, typhoid fever
• Acinetobacter baumannii - opportunistic, wounds, UTI, pneumonia
• Neisseria gonorrhoeae - gonorrhoea

Question 6

Major AB resistant bacterial pathogens - gram positive

Answer 6

• Staphylococcus aureus (MRSA, VISA) - wound and skin infection, pneumonia, septicaemia, infective endocarditis
• Streptococcus pneumoniae - pneumonia, septicaemia
• Clostridium difficile - pseudomembranous colitis, antibiotic-associated diarrhoea
• Enterococcus spp. (VRE) - UTI, bacteraemia, infective endocarditis
• Mycobacterium tuberculosis - tuberculosis

Question 7

Protonsil

Answer 7

• sulphonamide antibiotic
• bacteriostatic
• synthetic
• e.g. sulphamethoxazole, sometimes used together with Trimethoprim
• uses: treat UTIs, RTIs, bacteraemia and prophylaxis for HIV+ individuals
• becoming more common due to resistance to other antimicrobials, despite some host toxicity
• only against gram positive bacteria

Question 8

Beta-lactams

Answer 8

• interfere with the synthesis of the peptidoglycan component of the bacterial cell wall
• e.g. penicillin, methicillin
• binds to penicillin-binding proteins (PBPs)
• PBPs catalyse a number of steps in the synthesis of peptidoglycan

Question 9

Aminoglycosides

Answer 9

• e.g. gentamicin, streptomycin
• bactericidal
• targets protein synthesis (30S ribosomal subunit), RNA proofreading and causes damage to cell membranes
• toxicity has limited use, but resistance to other antibiotics has led to increasing use

Question 10

Rifampicin

Answer 10

• bactericidal
• targets RpoB subunit of RNA polymerase
• spontaneous resistance is frequent
• makes secretions go orange/red - affects compliance

Question 11

Vancomycin

Answer 11

• bactericidal
• targets lipid II component of cell wall biosynthesis, as well as wall crosslinking via D-ala residues
• toxicity has limited use, but resistance to other antibiotics has led to increasing use

Question 12

Linezolid

Answer 12

• bacteriostatic
• inhibits initiation of protein synthesis by binding to 50S rRNA subunit
• gram positive spectrum of activity

Question 13

Daptomycin

Answer 13

• bactericidal
• targets bacterial cell membrane
• gram positive spectrum of activity
• toxicity limits dose

Question 14

Macrolides

Answer 14

• e.g. erythromycin, azithromycin
• gram-positive and some gram-negative infections
• targets 50S ribosomal subunit preventing amino-acyl transfer and thus truncation/production of polypeptides

Question 15

Quinolones

Answer 15

• synthetic, broad spectrum, bactericidal
• target DNA gyrase in Gm-ve and topoisomerase in Gm+ve

Question 16

Selective toxicity

Answer 16

• large number of differences between mammals and bacteria result in multiple targets for AB therapy - selective toxicity

Question 17

Mechanisms of antibiotic resistance - altered target site

Answer 17

• can arise via acquisition of alternative gene / gene that encodes a target-modifying enzyme
• e.g. MRSA encodes an alternative PBP with low affinity for beta-lactams
• e.g. Streptococcus pneumoniae resistance to erythromycin occurs via the acquisition of the ‘erm gene’ - encodes an enzyme which methylates the AB target site in the 50S ribosomal subunit

Question 18

Mechanisms of antibiotic resistance - inactivation of antibiotic

Answer 18

• enzymatic degradation or alteration, rendering antibiotic ineffective
• e.g. beta-lactamase (bla) and chloramphenicol acetyl-transferase (cat)
• ESBL and NDM-1 are examples of broad-spectrum beta-lactamases

Question 19

Mechanisms of antibiotic resistance - altered metabolism

Answer 19

• increased production of enzyme substrate can outcompete AB by competitive inhibition (e.g. increased production of PABA confers resistance to sulfonamides)
• alternatively, bacteria switch to other metabolic pathways, reducing requirement for PABA

Question 20

Mechanisms of antibiotic resistance - decreased drug accumulation

Answer 20

• reduced penetration of AB into bacterial cell (permeability) and/or increased efflux of AB out of the cell - drug does not reach concentrations required to be effective

Question 21

Multiple resistance systems can coexist

Answer 21

antibiotic, resistance mechanism, acquired

• penicillin, penicillinases, plasmid-transformation
• penicillin, target site modification, point mutation
• tetracycline, efflux pump, plasmid-conjugation
• quinolone, target site modification, point mutation
• cefotaxime, target site modification, point mutation
• spectinomycin, target site modification, point mutation

Question 22

Sources of antibiotic resistant genes

Answer 22

• plasmids - extra chromosomal circular DNA, often multiple copy, often carry multiple AB res genes - selection for one maintains resistance to all
• transposons - integrate into chromosomal DNA - allow transfer of genes from plasmid to chromosome and vice versa
• naked DNA - DNA from dead bacteria released into environment
• genes responsible for conferring AB resistance can be shared between bacteria via several different mechanisms - transformation (uptake of extracellular DNA), conjugation (pilus-mediated DNA transfer), transduction (phage-mediated DNA transfer)

Question 23

Non-genetic mechanisms of resistance / treatment failure

Answer 23

• biofilm - matrix-encased communities of bacteria that are highly drug tolerant
• intracellular location - some bacteria persist within human cells, making it hard for AB to access
• slow growth - if bacteria aren’t using many processes to replicate, it’s hard for AB to inhibit
• spores - impermeability of the spore coat is thought to be responsible for resistance
• persisters - dormant organisms that aren’t using any processes that AB inhibit so not killed by them - do not grow in the presence of antibiotics

Other reasons for treatment failure:

• inappropriate choice for organism
• poor penetration of AB into target site
• inappropriate dose (half-life)
• inappropriate administration (oral vs IV)
• presence of AB resistance within commensal flora e.g. secretion of beta-lactamase

Question 24

Hospital acquired infections (HAIs)

Answer 24

• large numbers of infected people receiving high doses of antibiotics = strong selective pressure for AB resistance
• MRSA
• VISA (vancomycin-insensitive S. aureus)
• Clostridium difficile
• VRE (vancomycin-resistant enterococci)
• E. coli (ESBL/NDM-1)
• P. aeruginosa
• Acineterbacter baumannii
• Stenotrophomonas maltophilia

Question 25

Risk factors for HAI

Answer 25

- high number of ill people (immunosuppression) - crowded wards - presence of pathogens - broken skin - surgical wound/IV catheter - indwelling devices - intubation - AB therapy may suppress normal flora - transmission by staff - contact with multiple patients

Question 26

Addressing and overcoming resistance

Answer 26

- prescribing strategies - tighter controls, temporary withdrawal of certain classes, restriction of ABs for certain serious infections - reduce use of broad-spectrum ABs - quicker identification of infections caused by resistant strains - combination therapy - knowledge of local strains / resistance patterns Overcoming resistance: - modification of existing mechanisms to e.g. prevent cleavage (beta-lactams) or enhance efficacy (e.g. methicillin) - combinations of AB + inhibitor of e.g. beta-lactamase e.g. augmentin - however this is a reactive approach in response to emergency

Question 27

Measuring resistance

Answer 27

1. swabs are typically streaked out onto diagnostic agar to identify the causative organism 2. once identified, the pathogen streaked over a plate and then overlaid with AB-containing test strips or discs - other approaches include broth micro-dilution and PCR detection of resistance genes - measurements are made in vitro = may not fully reflect the situation in vivo

Question 28

What are the three classes of conditions fungi cause in humans?

Answer 28

- allergy - allergic reactions to fungal products e.g. allergic bronchopulmonary aspergillosis (ABPA) - mycotoxicoses - ingestion of fungi and their toxic products e.g. aflatoxin - mycoses - superficial, subcutaneous or systemic colonisation, invasion and destruction of human tissue

Question 29

What are the targets for antifungal therapy?

Answer 29

- cell membrane - fungi use principally ergosterol instead of cholesterol - DNA synthesis - some compounds may be selectively activated by fungi, arresting DNA synthesis - cell wall - unlike mammal cells, fungi have a cell wall