Lecture 2: Introduction to Superbugs

Question 1

What are superdrugs?

Answer 1

Superbugs are bacterial strains that have become resistant to multiple antibiotics used for treatment (Multi-Drug Resistant - MDR).

Question 2

Give some examples of superbugs

Answer 2

• Staphylococcus aureus (MRSA)
• Escherichia coli
• Acinetobacter spp.
• Klebsiella pneumoniae
• Pseudomonas aeruginosa,
• Mycobacterium tuberculosis

Question 3

Historical Insight

Answer 3

Alexander Fleming (1945 Nobel Lecture) warned that inappropriate antibiotic use could lead to resistance by allowing bacteria to “learn” to resist penicillin.

Question 4

Describe the timeline of resistance development of S.aureus to penicllin

Answer 4

• 1940s-1950s: Penicillin used to treat Staphylococcus aureus.
• 1944: First reported case of penicillin-resistant S.aureus
• 1959: Introduction of Methicillin to treat penicillin-resistant S.aureus
• 1961: British scientists reported the first case of MRSA
• 1968: First reported human case of MRSA in the United States
• 2002: First documented case of VRSA (vancomycin-resistant Staphylococcus aureus) in the United States

Question 5

What does VRSA stand for?

Answer 5

vancomycin-resistant Staphylococcus aureus

Question 6

What did resistance to vancomycin first develop in?

Answer 6

Resistance to vancomycin developed in Enterococcus and it is now quite common in Enterococcus

Question 7

Is it true that VRSA strains are still quite rare?

Answer 7

Yes

Question 8

Describe the mechanism of antibiotic resistance

Answer 8

• Resistance arises due to exposure to antibiotics, especially at sub-therapeutic doses. This selects for bacteria with mutated key genes (e.g, up-regulation of β-lactamases; down-regulation of porins; up-regulation of efflux pumps…)
• These genetic information will pass from one generation to the next within a family via Vertical Gene Transfer.
• The genetic information can also spread across several bacterial species- Horizontal Gene Transfer via the plasmids

Question 9

What are the anthropogenic drivers of antibiotic resistance?

Answer 9

• Farming practices
• Horticultural practices
• Veterinary practices
• Healthcare practices

Question 10

How do farming practices drive antibiotic resistance?

Answer 10

• Use of antibiotics as animal growth promoters
• Many animals routinely fed (subtherapeutic) doses of β-lactams, erythromycin and tetracycline as growth promoters

Question 11

How do horticultural practices drive antibiotic resistance?

Answer 11

• Antibiotics sprayed on crops, to reduce bacterial growth
• E.g, Californian soft fruit are routinely sprayed with tetracycline to reduce bacterial growth

Question 12

How do verterinary practices drive antibiotic resistance?

Answer 12

Over-prescription of antibiotics for treatment of pets

Question 13

How do healthcare practices contribute to antibiotic resistance?

Answer 13

• Overprescription by GPs, especially for viral infections like respiratory tract infections.
• Unregulated access to antibiotics in developing countries.

Question 14

How did Covid 19 contribute to increased antibiotic resistance?

Answer 14

Lack of clear protocols led to increased inappropriate antibiotic use.

Question 15

What are the 2 types of resistance mechanisms for antibiotic resistance?

Answer 15

Intrinsic Antibiotic Resistance

&

Acquired Antibiotic Resistance

Question 16

What is intrinsic resistance?

Answer 16

Naturally occurring, e.g., Gram-negative bacteria with double membranes that inhibit drug entry.

Question 17

What is extrinsic resistance?

Answer 17

Results from genetic changes like acquiring β-lactamase genes or efflux pumps that expel antibiotics.

Question 18

Describe non-specific antibiotic resistance

Answer 18

Affects multiple antibiotics via mechanisms such as:

• Efflux pumps (removing antibiotics).
• Reduced membrane porins (limiting drug entry).

Question 19

Describe specific antibiotic resistance

Answer 19

• Targets a particular antibiotic or class. Examples:
• Production of enzymes like β-lactamases.
• Mutations at antibiotic target sites (e.g., ribosomes).(to resist macrolides.)

Question 20

Describe the ways in which resistance to beta-lactam antibiotics can occur?

Answer 20

① mutation of PBP, lowering the affinity for penicillins, etc

② down-regulation of porins in Gram-negative bacteria;

③ acquisition of β-lactamase- ESBL (extended spectrum β-lactamase);

④ up-regulation of efflux pumps

Question 21

Describe the ways in which resistance to quinolones may occur

Answer 21

① Mutations in gyrase weaken quinolone-enzyme interactions

② Plasmid – encoded Qnr proteins decrease topoisomerase–DNA binding

③ A plasmid-encoded enzyme acetylates ciprofloxacin, decreasing its effectiveness.

④ Plasmid-encoded efflux pumps decrease the concentration of quinolones in the cell.

Question 22

Describe how resistance to Aminoglycosides may occur

Answer 22

• Aminoglycosides can be modified by acetyltransferases
• Methylation of the 16S rRNA by ribosomal methyltransferase prevent Aminoglycosides from binding to this target

Question 23

Which multi-drug -resistant bacteria is a rising threat?

Answer 23

• Klebsiella pneumoniae
• K. pneumoniae: causes the disease Klebsiella pneumonia, with destructive changes to human lungs, inflammation and haemorrhage that sometimes produces a thick, bloody, mucoid sputum (currant jelly sputum).

Question 24

Where is there a high prevalence of Klebsiella pneumoniae

Answer 24

High prevalence in Greece and Italy (>50% of isolates resistant in 2010).

Question 25

What is typing important for?

Answer 25

- Necessary for controlling outbreaks and tracking the spread of resistance. (ie k. pneuomniae) - Helps in epidemiological studies and implementing control measures.

Question 26

Describe typing with gram stain

Answer 26

Gram stain checks for peptidoglycan, a substance in bacterial cell walls. It's a useful test to quickly identify types of bacteria. But it doesn’t work for all bacteria — some don’t show up clearly. Some bacteria give mixed results (called Gram-variable). Others don’t react at all (called Gram-indeterminate). The main problem: it can't tell the exact species — just general types (Gram-positive or Gram-negative).

Question 27

What are the phenotypic methods of typping?

Answer 27

- Biotyping - Antibiotyping - Phage Typing - Serotyping

Question 28

Describe biotyping

Answer 28

- Examines growth profiles, metabolic activities, and colony morphology./ a method of identifying and classifying organisms based on their metabolic activities, environmental tolerances, and other characteristics./The identification of different bacteria types based on reaction to biochemical tests. - Advantages: Reproducible, easy to perform. - Disadvantages: Limited discriminatory power due to variation in gene expression and point mutation alter metabolic activities

Question 29

What is Antibiotyping?

Answer 29

- strain discrimination on the basis of antibiotic resistance. Comparison of susceptibility of different isolates to a set of antibiotics. Isolates differing in their susceptibilities are considered as different strains. - Advantages: almost all strains are typeable. Reproducible. Easy to perform and interpret - Disadvantages: acquisition of antibiotic determinants, point mutations and gene expression changes can alter patterns of antibiotic resistance quickly, reducing discriminatory power

Question 30

What is phage typing?

Answer 30

-strain discrimination on the basis of resistance to various bacteriophages. - Strains are characterised by their resistance or susceptibility to a standard set of bacteriophages and relies on the presence or absence of particular receptors on the bacterial surface that are used by the virus. Advantages: Fairly reproducible, good discriminatory power and easy to interpret. Disadvantages: This technique requires maintenance of biologically active ‘phages and hence is available only at reference centres. Even for experienced workers, the technique is time-consuming. Many strains are non-typeable.

Question 31

Define serotypoing

Answer 31

- Differentiates strains by antigenic properties (e.g., lipopolysaccharides, flagella). - Strains differentiated by antigenic differences known as 'serotypes'. - Common for Salmonella (over 2,600 serotypes identified). - Disadvantages: Technically demanding, cross-reactivity issues.

Question 32

Describe the genotypic methods of typing

Answer 32

- DNA-based techniques like: - RAPD (Random Amplification of Polymorphic DNA). - PFGE (Pulsed Field Gel Electrophoresis). - MLST (Multilocus Sequence Typing). - Provide superior strain discrimination and reveal genetic relationships.

Question 33

Summary

Answer 33

- There are global present and rising threats from multi-drug resistant pathogenic bacteria - Inappropriate use of antibiotics is the primary cause of the development and spread of antibiotic resistance. - We need accurate typing methods to examine the natural history of spread of a new variant and to apply control measures - Phenotyping methods take some way towards identification, but nucleic acid techniques should show superior strain discrimination.

Question 34

Exam Preparation Focus on: Antibiotic resistance mechanisms. Examples of superbugs. Spread of resistance via gene transfer. Drivers of resistance spread. Principles of typing methods.

Answer 34

Exam Preparation Focus on: Antibiotic resistance mechanisms. Examples of superbugs. Spread of resistance via gene transfer. Drivers of resistance spread. Principles of typing methods.

Question 35

phenotypic vs genotypic typing

Answer 35

- Phenotypic methods are easy but lack precision. - Genotypic methods provide detailed information but are resource-intensive.

Question 36

Is it true that both gram negative and gram positive bacteria can have efflux pumps?

Answer 36

Yes

Question 37

What are the 2 different categories of typing?

Answer 37

- Phenotypic and genetic