Mini 3 Practice Questions

B and D. These are both possible—E. coli is Gram-negative, so can modify outer membrane proteins to decrease permeability, and also expresses efflux pumps which can be quite non-specific in terms of what they pump out. The other mechanisms would likely be limited to a single or related classes of antibiotic, and/or don’t fit with these antibiotics.

B. All resistance in Mycobacterium tuberculosis seen to date is due to point mutation. In this case, it can occur either in the gene for the antibiotic target (inhA), or in the gene for a catalase that activates isoniazid to its bactericidal form (katG). What other class of pathogen is also restricted to point mutation to develop resistance?

List two organisms that have relatively impermeable outer membranes. This is one, Acinetobacter is the other one.
Reduced uptake requires an outer membrane (where the porins live). The only organism with an outer membrane on this list is Pseudomonas. It and Acinetobacter have many porins to choose from, which allows them to make their outer membranes more selective than many other bacteria.

C. Antibiograms compile AST data to allow local resistance trends to be seen and increase the odds of picking an effective empiric (i.e. immediate, before getting specific AST results) treatment.

Study question: How does the exotoxin produced by this organism work? How is this organism cultured? What are its characteristics?

A. The other choices all have a component referring to a different target or require a structure not present in this organism.

Bactericidal drugs kill bacteria, giving the blue line.
Bacteriostatic antibiotics stop growth without killing, giving the green line.
Resistance allows growth to continue in the presence of antibiotic, giving the red line.
Tolerance would cause a bactericidal drug to act in a bacteriostatic way, giving the green line.

Conjugation is the most common mechanism of resistance due to acquired means. HFr strains (i.e. able to conjugate chromosomal genes) are much less common than F+ strains. Transformation is limited to naturally competent species, so this could be a good answer if, for example, Streptococcus or Neisseria is implied. Transduction is not a common mechanism for resistance gene transfer. Point mutation is a very common mechanism of resistance, but is a rare event and thus unlikely to occur in multiple, unrelated genes at the same time.

MRSA has acquired the PBP2a gene, which encodes a transglycosylase/transpeptidase enzyme that is insensitive the meth/naf/oxa. Most staphylococci do express penicillinases, but these will not degrade the penicillinase-resistant penicillins. Point mutation in the PBP could give this type of resistance, but it is not commonly seen. ESBLs (Extended Spectrum Beta Lactamases) can also degrade these drugs, but are not found in staphylococci. Altered PG monomer is the mechanism for vancomycin resistance (see further down in the lecture), and ribosome methylation would not provide beta-lactam resistance (wrong target).

Study question: What other mechanisms of resistance could give a similar phenotype?

B. This Streptococcus pyogenes isolate is displaying MLSB resistance, which stands for macrolide, lincosamide and streptogramin B resistance. It is most likely caused by acquisition of an Erm methyltransferase, which methylates a base in the 23S rRNA gene. Interestingly, this type of resistance in this bacterial species is not very common in the US, but is more so in some European countries.

C. Vancomycin is unable to cross the outer membrane and is thus inactive against Gram-negative bacteria such as Escherichia. This is innate because the outer membrane is a fundamental part of the cell, it does not change it or express it differently in any way to resist the action of Vn.

C. S. aureus became resistant to penicillins by acquiring penicillinases, and then acquired the mecA gene to become MRSA. The intermediate Van resistance shown here is actually due to a mechanism distinct from that seen in VRSA. This mechanism is not terribly well understood, but involves a thickening of the cell wall layer and possibly reduced cross-linking. This means that there are more D-ala-D-ala tails present throughout the wall, which bind the drug as it penetrates and prevent it from getting to the cell surface, where the new monomers appear and are incorporated. We didn’t look at this particular mechanism in class, but I hope you realize that VRSA displays higher-level resistance (definitely in the R range, usually up at the higher end).

All of these are possible to some extent. Downregulation could occur via a mutation in a promoter region gene for a regulatory protein, which would be in a similar range of ‘likelihood’ as mutations within the resistance gene. Transposons moving into a resistance gene would reverse resistance, but again, is unlikely. Recombination implies a source of a defective resistance gene, and recombination is also not likely. The MOST likely option is loss of plasmid. Since plasmids do not partition equally upon cell division, daughter cells with no plasmid do arise. In the absence of selective pressure, these plasmid-free cells will replicate better than those carrying plasmid (less DNA and proteins to make, more energy to devote to growth), and will become the predominant/only cell type in the population over successive generations.

The organism here is Staphylococcus. Most staphylococci are resistant to penicillins via penicillinase. MRSA/MRSE are also resistant to nafcillin/oxacillin/methicillin, and comprise the majority of clinical staphylococcal isolates in the US. Relatively few staphylococcal isolates are resistant to the other antibiotics in this list (at the moment…).

Study question: What is the causative agent? How does the Etest work?

C. Tobramycin is an aminoglycoside, and covalent modification with a small molecule (in this case, adenylation) via an acquired transferase enzyme is an important mechanism of resistance to this class of antibiotic. The causative agent here is Pseudomonas aeruginosa—remember its importance to CF prognosis? How does this organism cause disease progression in these patients?

C. The important concept here is selective pressure. This could mean selecting for mutations or acquired elements; however, in an infection caused by a single organism, there is no source of acquired DNA.

F. At the heart of it, the thing driving resistance is bacterial survival, and use of antibiotics is the pressure that selects for resistance. Anything that reduces antibiotic use (either totally, by preventing infections, or of specific drugs) or allows bacterial survival without disease (effectively making a pathogen a commensal) will reduce this pressure.

D. The organism being described is Enterococcus, specifically vancomycin-resistant Enterococcus (VRE). The resistance mechanism in this case is acquisition of a van element, which encodes proteins resulting in a change in the peptidoglycan monomer structure. This altered structure is not efficiently bound by vancomycin.

E. The van element is on a transposon, which is thought to have been present on a conjugative plasmid and acquired via conjugation. Most acquired antibiotic resistance is thought to have been via conjugation, although transformation is a significant route in naturally competent organisms. Make sure you can explain these processes!

C. The mecA gene encodes PBP2’ (PBP2a), which is a transglycosylase/transpeptidase enzyme that is insensitive to penicillins and cephalosporins.

A. The most common mechanism of tetracycline resistance (in the absence of any contravening evidence) is efflux. Efflux pumps are transmembrane proteins that require either energy from the proton motive force or ATP to move the antibiotic from a volume of low concentration (inside the cell) to high concentration (outside the cell). Other mechanisms definitely exist, such as that described in E or B, point mutations in ribosomal components, as well as a concept called ‘ribosome protection’ which we did not discuss in class but is, in fact, the 2nd most common mechanism. This involves proteins which bind the ribosome and prevent tetracycline binding.

A. Make sure you know what the organisms are, and realize that this doesn’t cover all of them
(far from it).
 Enterococcus VRE
 Staphylococcus- MRSA, MRSE, b-lactamases
 Klebsiella - ESBL, carbapenems
 Acinetobacter - ESBL, efflux, OMPs, etc.
 Pseudomonas - ESBL, efflux, OMPs, etc.
 ESBL - G- rods
 Tuberculosis - MDR and XDR
Streptococcus pneumoniae and Neisseria gonorrhoeae are also problematic (see notes)

D. Kirby Bauer is also known as the disc diffusion method. Make sure you know what a zone of inhibition is, why it is caused, and how to interpret the measurement from this test. Etest uses zones of inhibition, but does not require measurement of diameters (the MIC is right on the strip).

E. Breakpoints for antibiotic susceptibility testing are based on MIC, not MBC values. All of the other choices are used to help make in vitro breakpoints as predictive as possible for clinical use.

D. The antibiogram tells us that 63% of viridans streptococci are susceptible to erythromycin. This is a highly integrated question, so good if you got it on the first try!

TB and viral resistance are always due to point mutations. Escherichia, being a Gram-negative, is intrinsically resistant to vancomycin (the drug is large and hydrophobic, and is unable to cross the outer membrane). Aminoglycoside resistance is due to a transferase enzyme (of some sort), which must be acquired from somewhere.