Antimicrobials and Discovery of Resistance (8-13)

Question 1

What are biocides?

Answer 1

Disinfectants → products that are used on inanimate objects or surfaces
Antiseptics → products that destroy or inhibit the growth of microorganisms in or on living tissue
Sterilisation → physical or chemical process that completely destroys or removes all microbial life, including spores

Question 2

What is an antibiotic?

Answer 2

Low molecular substance often produced by a microorganism that at a low concentration inhibits or kills other bacteria

Question 3

What is an antimicrobial?

Answer 3

Any substance of natural, semisynthetic or synthetic origin that kills or inhibits the growth of microorganisms but causes little or no damage to the host

Question 4

What is the difference between antibiotic resistance and antimicrobial resistance?

Answer 4

Antibiotic resistance → occurs when bacteria change in response to the use of these medicines
Antimicrobial resistance → is a border term, encompassing resistance to drugs to treat infections caused by other microbes as well as parasites (e.g. malaria), viruses (e.g. HIV) and fungi (e.g. Candida)

Question 5

What are the different sources of antimicrobials?

Answer 5

Plant-based → essential oils
Metal-based → copper, silver
Nanotech-based → antimicrobial peptides, fullerenes, nanotubes, nanoparticles
Animal-based → escapin (sea hare), chitosan (shells), snake venom
Microbe-based → antibiotics, bacteriocins, bacteriophage eddolysions

Question 6

Whats an example of a plant-based antimicrobial?

Answer 6

Cephaelis ipecacuanha
→ found in Central America
→ ground up roots are used as an emetic: throwing up, not the benfitical effect but the antimicrobial effect
→ flawed logic but still worked
→ modern medicine: extract antimicrobial agent
→ its not safe, no longer in legal use

Question 7

What is curcumin?

Answer 7

Yellow compound derived from turmeric
→ from plant Curcuma longa
→ natural phenolic compound
→ potent anti-tumor, anti-inflammatory, anti-oxidant, anti-microbial
→ synergistic (works better combined) effects with silver ions and antibiotics

Question 8

What is the antimicrobial affect of silver?

Answer 8

Synergistic effects with:
→ silver ions, silver nanoparticles, silver ions with other metal ions, silver and antibiotics

→ can penetrate bacterial cell walls, cause DNA damage, effect proteins

Question 9

What is escapin?

Answer 9

Animal-based antimicrobial from sea hare
→ L-amino acid oxidase, flavin cofactor dependant
→ oxidises L-arginine and L-lysine to produce H2O2
→ bacteriostatic in minimal media
→ bactericidal in rich media
→ can also inhibit growth of yeast and fungi, with different efficacies

Question 10

What is the mode of action of L-amino acid oxidases?

Answer 10

Converts L-amimo acids (+H2O + O2) to pyretic acid and hydrogen peroxide H2O2
→ intracellular conc of H2O2 is tightly controlled and assumed to vary between 1 and 700nM
→ intracellular steady-state concentrations of H2O2 above 1 uM are considered to cause oxidative stress inducing growth arrest and cell death

Question 11

What is a biofilm?

Answer 11

An assembly of microbial cells associated with a surface, and enclosed in an extracellular matrix made principally of polysaccharides
→ provides protection for colony survival
→ allows bacteria to become tolerant to antibiotics/antimicrobials and generates resistance

Question 12

What is the architecture of bacteria matrix?

Answer 12

Provides stability
Has pores and channels
Fills space between cells

Question 13

Why are gram-negative bacteria resistant to many antibiotics?

Answer 13

Gram-negative bacteria are intrinsically resistant to many antibiotics due to
→ double-membrane structure that makes the cellular envelope relatively impermeable

Question 14

What are beta-lactam antibiotics

Answer 14

Antibiotics that inhibit cell wall synthesis
→ includes: penicillins, cephalosporins, cephamycins, monobactams…

Question 15

What do beta-lactam antibiotics target?

Answer 15

Penicillin-binding proteins
→ DD-transpeptidases that make cross-linked (peptide bonds) between D-amino acid residues in sugar-linked pentane-tides in bacteria cell walls
→ essential for bacteria cell wall synthesis

Question 16

How do beta-lactams work?

Answer 16

The reactive beta-lactam ring bind the active site of the transpeptidase
→ permanently inactivating the PBP (penicillin-binding proteins)
→ cell wall cross-linkage ceases

Question 17

How can resistance to beta-lactam antibiotics occur?

Answer 17

1. mutation of penicillin-binding proteins, lowers the affinity for penicillins
2. down-regulation of porin in gram -ve bacteria
3. acquisition of B-lactamase
4. up-regulation of efflux pumps

Question 18

What are antibiotics that inhibit nucleus acid synthesis?

Answer 18

Quinolone antibiotics
→ ciprofloxacin, ofloxacin, levofloxacin, nalidixic acid

Question 19

What is the target for quinolone antibiotics?

Answer 19

Target topoisomerases
→ regulate supercoiling in DNA synthesis and RNA synthesis (type II convert +ve over-wound into -ve under-wound)
→ interferes with nucleic acid synthesis by binding to topoisomerases

Question 20

How do ciprofloxacin (and other quinolone antibiotics) work?

Answer 20

Convert their target type II topoisomerases (gyrase and topoisomerase IV) into toxic enzymes that fragment the bacterial chromosome
→ gyrase can still make double stranded cuts but are hard to repair as they can’t be ligated by topo II
→ ultimately cause cell death

Question 21

How are bacteria resistant to quinolones?

Answer 21

1. mutations in gyrase and topo IV weaken quinolone-enzyme interactions
2. plasmid-encoded Qnr proteins decrease topoisomerase-DNA binding
3. palms-encoded enzyme acetylates ciprofloxacin, decreasing its effectiveness
4. plasmid-encoded efflux pumps decrease conc of quinolone in the cell

Question 22

What are antibiotics that inhibit protein synthesis?

Answer 22

Macrolides → effect translocation
Chloramphenicol → binds to the 50S ribosomal subunit - inhibits formation of peptide bonds
Tetracycline → binds to the 30S subunit - interferes with the binding of tRNA to the ribosomal complex
Aminoglycosides → binds to the 30S subunit - causes codon misreading

Question 23

How do aminoglycosides work?

Answer 23

Contain amino-sugar structures
→ display activity against gram -ve aerobes
→ inhibit protein synthesis by high affinity binding to the A-site on the 16S ribosomal RNA of the 30S ribosome
→ causes codon misreading - misincorporation on amino acids (can midfield proteins)
→ affects membranes - leads to rapid uptake of drug, increased inhibition of protein synthesis

Question 24

What do aminoglycosides have synergism with?

Answer 24

Antibiotics like beta-lactams
→ allows greater penetration of aminoglycosies at low dosages
→ all aminoglycosides are rapidly bactericidal to gram-ve in aerobic conditions

Question 25

How can bacteria be resistant to aminoglycosides?

Answer 25

Inactivation → can be modified by acetyltransferase
Target site modification → methylation of the 16S rRNA prevents aminoglycosides from binding to the target
Decreased influx/active efflux

Question 26

What are nosocomial infections?

Answer 26

An infection developing in a patient as a result of healthcare contact
→ had no signs of infection within first 48hours
→ important measure of quality of cleanliness and risk factors of healthcare services

Question 27

What are non-nosocomial infections?

Answer 27

An infection acquired outside the healthcare setting
→ infections diagnosed within 48 hours of admission

Question 28

What are the 5 main types of nosocomial infections?

Answer 28

Central line associated bloodstream infections
Catheter associated UTIs
Nosocomial pneumonia - ventilator associated
Surgical site infections
Gastrointestinal infection
→ mostly associated with a procedure - increased change of bacterial exposure

Question 29

What are some nosocomial pathogens?

Answer 29

Bacterial, fungi, viruses
→ bacterial: Pseudomonas aeruginosa, Staphylococcus aureus, E.coli, Clostridium difficile, Klebsiella spp
→ viral: covid 19, norovirus

Question 30

What are the risk factors for nosocomial infection?

Answer 30

Environment → poor hygienic conditions (inadequate waste disposal), covid - ventilation, overcrowding, lacking PPE
Susceptibility → immunosuppression, health age, age, use of medical devices, drug treatment, length of stay
Unawareness → improper injection techniques, poor knowledge of infection control

Question 31

How can nosocomial infection be prevented?

Answer 31

Environment hygiene
Hand hygiene
PPE
Safe disposal of sharps
Asepsis

Question 32

What increased the risk of hospital acquired C. diff infections?

Answer 32

Exposure to antibiotic → disrupt normal gut flora, C.diff quickly established (resistant; has selective advantage)
Exposure to other patients

Question 33

What are the risk factors for C. diff infections?

Answer 33

Antibiotics → significant: fluoroquinalones, cephalosporins
Gastric acid suppressants → protein pump inhibitors associated, links to disruption of microbiota
Co-morbidities → link o inflammatory bowel disease, chronic kidney disease, immunodeficiency and solid organ transplants

Question 34

How can Klebsiella spp. be transmitted?

Answer 34

Person-to-person contact, or less commonly contamination of environment
Exposure when on ventilators, intravenous catheters or wounds

Question 35

How can infection in healthcare settings drive emergent of resistance?

Answer 35

Prescriptions and over prescriptions → in primary healthcare settings drives selection pressure for resistance
Spread and longevity → of injection (i.e. in ill patients) increases opportunity to become resistant
Overlap of pathogens → genetic exchange

Question 36

How are community acquired C. diff infections transmitted?

Answer 36

Human contacts → children under 2 and their primary caregivers
Food → most presumptuous, retail meats
Animal → famers animals, some veterinary outbreaks
Environmental → soil bacteria contamination of dwellings, spores residing in kitchens from raw meat

Question 37

How is community acquired pneumonia controlled?

Answer 37

2 types of pneumococcal vaccines available
→ PCV given to all children, protects against 13 types of S. pneumoniae
→ PPV given to 65+ years, and high risk, protects against 23 types of S. pneumoniae
prevents more serious infections

Covid 19 → 2 vaccines licensed in Europe, boosters, masks, distancing, isolation

Question 38

What are superbugs?

Answer 38

Any strain of bacteria that’s become resistant to the antibiotics that are used to treat it
→ multi-drug resistance MDR
→ Staphylococcus aureus, E. coli, Klebsiella pneumoniae, Pseudomonas aeruginosa

Question 39

What are the mechanisms of resistance?

Answer 39

Exposure to antibiotics selects for bacteria:
→ with mutated key genes and/or their control systems (e.g. up-regulation and/or mutation of beta-lactamases)
→ with horizontally acquired antibiotic resistance determinants

Question 40

Why is Klesbsiella pneumoniae a rising threat?

Answer 40

It causes destructive changes to human lungs, inflammation and haemorrhage

Important in nosocomial infections → hospital-acquired, patients or staff

Increased 1999 to 2014, small increase 2014-2017 (sign of control)

Question 41

What are they anthropogenic (human activity) drivers of antibiotic resistance?

Answer 41

Farming practices → 13mil antibiotics used as growth promoters, routinely fed antibiotics

Horticultural practices USA → Californian soft fruit sprayed with tetracycline

Vetinary practises UK → over-prescription of antibiotics

Healthcare worldwide → over-prescription

Question 42

What does Gram staining involve?

Answer 42

Cells are heat fixed
→ stain with crystal violet
→ then iodine
→ ethanol removes crystal violet/iodine

Gram +ve → purple
Gram -ve → pink

Valuable diagnostic in clinical research settings, but not all can be classifies with this technique

Question 43

What is biotyping?

Answer 43

Strain discrimination by examining growth profiles on different substrates
→ metabolic activates, colony morphology, environmental tolerances compared

Adv: reproducible, easy to perform and interpret
Disadv: poor discriminatory power (mutations can alter metabolic activity)

Question 44

What is antibiogram typing?

Answer 44

Strain discrimination on the basis of antibiotic resistance
→ comparison of susceptibility of different isolates to a set of antibiotics

Adv: almost all strains are tapeable, reproducible
Disadv: acquisition of antibiotic determinants, point mutations and gene expression changes can alter pattern of antibiotics resistance

Question 45

What is phage-typing?

Answer 45

Strain discrimination on the basis of resistance to various bacteriophage
→ characterised by their resistance or susceptibility to a standard set of bacteriophages, relies on present or absent of particular receptors

Adv: fairly reproducible, good discriminatory power, easy to interpret

Disadv: required biologically active hoagies, time-consuming, many strains are non-typable

Question 46

What are bacteriophages?

Answer 46

Viruses that infect bacteria

Question 47

What is serotyping?

Answer 47

Strain discrimination on the basis of binding antibodies of non specificity to their cell surface antigens

Adv: most strains are to-able

Disadv: some are untypeable, technically demanding, poor discriminatory power

Question 48

Why do we need accurate typing methods?

Answer 48

To examine the natural history of spread of a new variant and apply control measures
→ bio typing takes us some way towards identification, but nucleic acid techniques show superior strain discrimination

Question 49

What is Pseudomonas aeruginosa?

Answer 49

A gram negative bacteria often found in water or soil
→ an opportunist pathogen that rarely affects healthy individuals (typically neutral but can cause disease when the body’s resistance is altered)

Question 50

What types of infection can Pseudomonas aeruginosa cause in high-risk patients?

Answer 50

Pneumonia → cystic fibrosis patients
Septic shock → neutropenic patients (low numbers of neutrophils)
UTI → catheterised patients
GI → premature infants, neutropenic caner patients
Skin/soft tissue → burns victims, wound infections, diabetics

Question 51

What are some Pseudomonas aeruginosa reservoirs?

Answer 51

Water sources → a problem for hospitals
Paws and hair samples
Contaminated oil spills

Question 52

What is DNA gel electrophoresis?

Answer 52

DNA is a negatively charged molecule
→ placed in agarose gel in an electric field, -ve DNA moves towards the positive electrode
→ gel matrix is difficult for large DNA fragments to move through, large fragments lag behind small fragments move rapidly

Separates DNA fragments on the basis of size

Question 53

What is pulsed field gel electrophoresis (PFGE)?

Answer 53

A variation of gel electrophoresis that introduces an alternating voltage gradients to improve the resolution of larger molecules
→ voltage periodically switched among 3 directions
→ takes longer than normal gel electrophoresis

Adv: good for analysing recent outbreaks, highly discriminative, faster than MLST

Disadv: many hours of work, very technical, results subjective

Question 54

What is multi locus sequence typing?

Answer 54

Typing of multiple genetic loci
→ isolated of microbial species are characterised by deriving DNA sequences of internal fragments of multiple genes
→ DNA amplified with PCR

Question 55

What are the non mechanisms of carbapenam resistance in P. aeruginosa?

Answer 55

1. down regulation of porins
2. acquisition of carbapenemases
3. up-regulation of efflux pumps

→ resistance phenotypes have spread to other gram -ve pathogens

Question 56

How has quinolone resistance arisen?

Answer 56

Quinolones inhibit DNA gyrase (topoisomerase II) and DNA topoisomerase IV

Arisen from mutations in
→ GyrA: mutated DNA gyrase resists quinolone binding
→ NfxB: globally dysregulated physiology that unregulated the efflux pumps

Question 57

What does coagulase test for?

Answer 57

Staphylococcus aureus
→ coagulase converts fibrinogen into fibrin
→ clumping of plasma is a positive result for coagulase

Question 58

What are the likely reasons for the reduction in MRSA cases in Europe?

Answer 58

Reducing the impact of risk factors
→ improved screening in hospitalised patients
→ better infection control practices
→ better barrier precautions

Increasing knowledge of environmental reservoirs and longevity of S. aureus
Improved knowledge of molecular mechanisms of resistance
Introduction of the antibiotic Vancomycin

Question 59

What are the reservoirs of MRSA?

Answer 59

Healthcare-associated MRSA
Livestock-associated MRSA
Community-associated MRSA
Fomite-associated MRSA

Question 60

Why is MRSA difficult to eradicate once in a hospital?

Answer 60

S. aureus is carried by patients and staff n the skin or intra-nasally, and can survive in the surfaces and dust of hospital environments
→ MRSA was demonstrated to survive in sterile goods packaging for more than 38 weeks
→ survival varies between strains

Question 61

What are some risk factors of nosocomial MRSA?

Answer 61

(increases the chance of developing the disease)
→ prolonged hospital stay
→ treatment with broad spectrum antibiotics
→ treatment in intensive care or a burns unit
→ proximity to another patients with MRSA
→ surgical wounds
→ infected people from the community entering hospitals
→ resistance to ethanol hand washes

hospital MRSA incidence has lowered worldwide

Question 62

What are some risk factors of community-associated MRSA?

Answer 62

Frequent skin-to-skin contact
→ sports participants
→ men who have sex with men
→ living in crowded conditions (e.g. inmates in prison, children in daycare, elderly in care)
→ having or touching cut/grazed skin

From fomites
→ sharing common personal items
→ touching contaminated surfaces

Others
→ being HIV positive
→ getting tattooed

Question 63

What are the key virulence factors of MRSA?

Answer 63

SCCmec = staphylococcal cassette chromosome mec
→ a mobile genetic element
→ includes meA gene - resistant to methicillin
→ gene spread via HGT

PVL = Panton-Valentine leukocidin
→ an exotoxin that stimulates apoptosis of granulocytes and monocytes
→ secretes alpha-toxin

Question 64

How has S. aureus penicillin resistance evolved?

Answer 64

1952: 72% of isolates were penicillin resistant
→ mechanism: acquisition of beta-lactamases

1960s: methicillin resistance - methicillin was introduced (similar to penicillin but acyl group prevents beta-lactamase activity)
→ low level resistance developed by: hyper production go beta-lactamase, modification of the target for penicillin (reduced affinty for methicillin)
→ high level resistance: acquisition of new PBP, encoded by mecA (low affinity fo methicillin)

Question 65

How is S. aureus resistant to multiple drugs?

Answer 65

Some strains shows multiple resistance to many antibiotics including: aminoglycosides, erythromycin, quinolones, tetracycline
→ mechanism: drug efflux pumps

Question 66

What is the antibiotic of choice for treating MRSA infections?

Answer 66

Vancomycin
→ though some strains are resistant or have reduced susceptibility to vancomycin
→ resistance evolved by: mutated peptide chains of peptidoglycan cannot bind vancomycin - cell walls can be synthesised

Question 67

What is MRSA?

Answer 67

‘superbug’ Methicillin-resistant Staphylococcus aureus
→ skin infection: red, swollen bump + fever