Antibacterial Therapy

Question 1

What is meant by time dependent and concentration dependent bactericidal activity

Provide examples of each type of antibiotic

Answer 1

• Time-dependent antibacterial refers to an antibiotic for which the plasma concentration of the unbound drug needs to remain at ~ 4 times the MIC for 50-80% of the dosing interval to be effective
  
  • Increased drug frequency or a CRI would increase the %T > MIC
  • Beta-lactams, cephalosporins, clindamycin
• Concentration dependent antibiotics achieve increased bactericidal activity as the ratio of drug concentration to MIC increases
  
  • Larger doses less frequently (to minimise adverse effects) improve the effects
  • INcreased dose - increased C_max:MIC
  • Aminoglycosides and fluoroquinolones

Question 2

Briefly describe the mechanism of action of the beta lactam antibiotics

Answer 2

• These drugs act by disrupting cell wall synthesis
• They are therefore most active against bacteria that are rapidly dividing and have a cell wall
• They block the final step (transpeptidation) of peptidoglycan synthesis by irreveribly binding to penicillin binding proteins
• Blocking cell wall synthesis stops bacterial division

Question 3

Explain the methods by which beta-lactam resistance can develop in bacterial populations

Answer 3

• Two major methods of resistance have been documented

1. Development of beta-lactamase
   
   • Beta-lactamase damages the beta-lactam ring, preventing binding to the PBP’s
   • This may be inherently present within the bacterial chromosome
   • B-lactamase genes can be transferred within the genetic material on plasmids
   • Use of a beta-lactam can induce beta-lactamase gene expression
2. Genetic alterations in the penicillin binding proteins
   
   • The altered PBP have a reduced affinity for beta-lactam binding
   • These bacteria include the methicillin resistant staphylococcus pseudomedius

Question 4

List and briefly detail the major differences between the various penicillins

Answer 4

1. Penicillin G
   
   • Original penicillin
   • poor spectrum of activity
   • Inactivated by beta-lactamase, gastric acid
2. Beta-lactamase resistant penicillins
   
   • methicillin
     
     • resistant to b-lactamase but inactivated by gastric acid
   • Oxacillin - resistant to gastric acid
     
     • less active when compared to pen G
3. Aminopenicillins
   
   • Susceptible to b-lactamase
   • Oral or parenteral
   • Wider spectrum against gram-negative aerobes
4. Carboxypenicillins
   
   • Carbenicillin and ticarcillin
   • Wider spectrum against gram negative aerobes
     
     • Especially pseudomonas
   • Susceptible to beta-lactamase, so gram positive spectrum is less than others
   • Ticarcillin clavulate can be useful for pseudomonas otitis
5. Uredopenicillins
   
   • Useful against pseudomonas and enterococcus
   • Not acid stable
   • Susceptible to beta-lactamase

Question 5

Describe the mechanism of action for cephalosporins

Answer 5

• The cephalosporins are a beta lactam antibiotic
• They are bactericidal by inhibiting cell wall synthesis
  
  • This results in osmotic fragility to the bacteria
• Bind to the PBP necessary for the final stage of transpeptidation in cell wall proteoglycan synthesis
• More effective than penicillins at penetrating the outer wall of gram-negative bacteria
• Less susceptible to beta-lactamase than penicillins
• Mostly excreted unchanged in the urine

Question 6

Briefly note the major differences between the different generations of cephalosporins

Answer 6

1. First generation. eg. Cephazolin, cephalexin
   
   • Active against gram positive aerobes and some gram negative aerobes (E coli, klebsiella, proteus)
   • Useful for gram positive skin and gram negative urinary tract infections
2. Second generation. eg. cephoxitin
   
   • Broader specturm cf. 1st gen.
     
     • Increased resistance to beta-lactamase
   • Increased efficacy against gram negative aerobes and anaerobes
3. Third generation. eg. cefixime, cefovecin (long acting)
   
   • Longer duration of activity
   • Undergo some hepatic transformation and biliary excretion
   • Marked activity against gram negative aerobes
   • Mostly administered parenterally
4. Fourth generation
   
   • Mostly resistant to b-lactamase
   • Similar to 3rd gen. not reliable against anaerobes

Question 7

Briefly outline the mechanism of action of the aminoglycoside antibiotic class

Answer 7

• Aminoglycosides interfere with the synthesis of bacterial protein
  
  • Binds to the membrane associated bacterial ribosome
  • Disturbs peptide elongation at the 30S ribosomal subunit
  • Specifically, impairs translational proof-reading
• Small, primarily basic, water soluble molecules
• Broad specturm of activity - requires oxygen to be active
  
  • gram negative and positive aerobic and facultative anaerobic bacteria
  • Particularly effective against aerobic gram negative bacilli
• Not effective against obligate anaerobes

Question 8

Note the novel and important pharmacokinetic properties of aminoglycosides with relevance to their antibacterial properties

Answer 8

• Poorly absorbed form the GIT
  
  • Parenteral administration is generally required to reach therapeutic concentrations
  • Neomycin can be used orally for local GIT effect
• Intravenous administration generally prefered due to concentration dependent mode of action
• Minimally bound to protein and poorly lipid soluble
  
  • Diffuse poorly into CNS, eyes, amniotic fluid, extracellular fluid compartments
• Smaller volume of distribution with obesity may facilitate a dosage reduction
• Rapidly excreted into the urine (unchanged) and urine levels are markedly elevated when compared to that of the plasma
• The drug accumulates within renal tubular cells and can be nephrotoxic
  
  • once daily high dose treatment lead to less accumulation

Question 9

Describe the mechanism of action of Vancomycin

Answer 9

• Vancomycin is a complex glycopeptide molecule
• A glycopeptide, vancomycin acts to inhibit cell wall synthesis
  
  • Primarily used for treatment of methicillin resistant staphylococcus aureus infection in humans
• Prevents NAG and NAM strand formation, the backbone components of the bacterial cell wall
  
  • Also prevents cross-linking of those strands that do form
• Poorly absorbed from the GIT but can be used orally to treat chronic Clostridial difficile colitis
• Wide distribution into most organs and does cross the BBB
• Largely excreted by the kidneys

Question 10

Provide notes on the mechanism of action of chloramphenicol

Answer 10

• Highly lipid soluble, broad spectrum antibiotic
• primarily bacteriostatic by inhibition of bacterial protein synthesis
• Prevents protein chain elongation by inhibition of peptidyl transferase activity of the bacterial ribosome
• Wide spectrum of activity including: Mycoplasma, Rickettsia, Ehrlichia, Anaplasma, Neorickettsia, Chlamydia, Bordatella, and most obligate anaerobes

Question 11

Briefly discuss the clinical indications for chloramphenicol

What are the potential precautions and side effects?

Answer 11

Clinical Indications:

• Can be used to treat many aerobic or anaerobic infections
• Preferred antibiotic for treating Salmonella and E coliinfections of the gut.
• Can be used for urinary tract infections
• Penetrates the cornea well and can be used to treat corneal lesions. Systemic use for intra-orbital lesions / deep corneal ulceration

Adverse effects / precautions:

• Can cause irreversible bone marrow failure / aplastic anaemia in humans
  
  • Idiosyncratic and not dose dependent
  • Major public health risk with drug handling
  • Banned for use in food-producing animals
• Reversible marrow damage reported in dogs and cats
  
  • More toxic in cats due to lack of glucuronidation pathway
• Salivation, nausea, depression, anorexia, dysphagia, vomiting and sporadic diarrhoea

Question 12

Describe the mechanism of action of the tetracycline antibiotic class

Answer 12

• Bacteriostatic
• Interfere with bacterial protein synthesis of the bacterial RNA
• Tetracyclines reversibly bind to both the 30S and 50S subunit of the bacterial ribosome
  
  • prevents introduction of new amino acids to the growing / nascent peptide chain
• Bacteria actively pump tetracyclines into the cytoplasm
  
  • While mammalian cells are relatively resistant to the effects
• Resistance can develop due to horizontal gene transfer for:
  
  • An efflux pump that eject tetracyclines from the cell
  • A ribosomal protection protein that dislodge the tetracycline and allow protein translation

Question 13

Document the clinical indications for tetracyclines in the treatment of dogs or cats

Answer 13

• Broad specturm of activity including aeroib and anaerobic gram positive and gram negative bacteria
• Effective against Mycoplasma, Rickettsia, Anaplasma, Chlamydia and some Protozoa
• Doxycycline is more lipid soluble giving it an increased activity agains anaerobes and facultative intracellular organisms such as Ehrlichia canis and Brucella canis
• Resistance can be present and is usually plasmid mediated
• Low dose continuous use may increase the risk of resistance development

Question 14

Discuss the clinical indications for the use of tetracyclines over and above their use as an antibiotic

Answer 14

• Tetracyclines inhibit the activity of neutral matrix metalloproteinases
  
  • Likely due to chelation of multivalent cations
• Anti-inflammatory activity in non-skeletal disorders, likely due to the same effect on metalloproteinases
• Have been shown to reduce the progression of osteoarthritis and cartilage degeneration by similar mechanism as above
• Can reduce periodontal disease in dogs - can be used as a polymer filling in periodontal pockets

Question 15

Describe the mechanism of action of the macrolide antibiotics

Answer 15

• Macrolides inhibit peptide formation by the 50S ribosomal subunit
  
  • Inhibit peptidyltransferase adding amino acids to the growing peptide chain (similar to chloramphenicol)
• Actively concentrated within leukocytes
  
  • Helps with transport and concentration at the site of inflammation
• Erythromycin may be poorly absorbed from the gut due to effects of gastric acid - enteric coating of tablets and taking them on an empty stomach can help absorption

Question 16

List the macrolide and lincosamide antibiotics used in veterinary medicine.

Briefly note the major indication for each

Answer 16

Macrolide:

• Erythromycin
  
  • As an alternative to penicillin
  • Campylobacter infection
• Azithromycin
  
  • Mycoplasma, Borrelia and Leptospira
  • Viral induced papillomas in dogs
• Tylosin
  
  • Gastrointestinal infections
• Clarithromycin
  
  • Toxoplasmosa and Mycobacterium

Lincosamide:

• Clindamycin
  
  • Primarily for gram positive aerobes and obligate anerobes
  • Oropharyngeal infection and abscess management

Question 17

Describe the clinical indications for the use of the macrolide antibiotics

Answer 17

• Primarily effective agains gram positive bacteria, Chlamydophyla, Mycoplasma, Helicobacter, Campylobacter and some Ricketsiae
• Slowly bactericidal antibiotic that has a poor correlation between drug concentration and rapidity of killing
  
  • Due to accumulation in neutrophils and macrophages
• Erythromycin is the first choice drug for Campylobacter and Legionella infection
  
  • Most often used as an alternative to Penicillins
• Tylosin can be indicated for clostridial enteritis, management of IBD / ARD and chronic colitis
• Erythromycin also has a pro-motility effect and helps to increase gastric emptying

Question 18

Describe the potential adverse effects of and contraindications for the use of macrolide antibiotics

Answer 18

• GI irritation is the most common adverse effect with vomiting, nausea and diarrhoea possible
• Some erythromycin formulations have been associated with hepatotoxicity and cholestasis
• Parenteral preparations are irritating at injection sites

Question 19

Note the mechanism of action, indications and potential adverse effects of clindamycin.

Answer 19

Mechanism of Action:

• Binds to the 50S subunit of the bacterial ribosome inhibiting protein synthesis - blocks peptidyl transferase
• Bactericidal at appropriate doses
• Concentrates within leukocytes - neutrophils and macrophages
• Widely distributed including into the bile, bone and cavitory fluids

Indications:

• Primarily for treatment of infection caused by gram positive aerobes and anaerobic bacteria
• Can also be used for protozoal infection such as Toxoplasma, Neospora, Babesia
• Osteomyelitis

Adverse effects / Contraindications:

• Relatively low toxicity
• GIT irritation, vomiting, diarrhoea
• Pseudomembranous colitis seen in humans and rarely in dogs
• Bitter taste can cause marked ptyalism in cats

Question 20

Describe the mechanism of action of the sulphonamide antibiotics

Answer 20

• Inhibit the bacterial synthesis of folic acid from para-aminobenzoic acid (PABA)
• Bacteriostatic
• Generally well absorbed and bound to plasma proteins
• Widely ditstributed to all tissues including pelural, peritoneal, synovial and ocular fluids.
• Readily enters the CNS and prostate
• Can be used to alter GIT flora without disrupting the balance of the microflora
  
  • Eg. sulphasalazine for colitis

Question 21

Note the clinical indications for the use of the sulfonamide antibiotics

Answer 21

• Bacteriostatic antibiotic
• Streptococcus, Bacillus, Corynebacterium, Nocardia, Campylobacter, Pasturella, Chlamydiae
• Note that disk diffusion susceptibility testing is not well correlated with in vivo susceptibility
• Primarily indicated for systemic infections or infection is specific locations such as the prostate or CNS
• Can also be used for gastrointestinal disease including colitis due to ability to reduce GIT bacterial load

Question 22

Describe the potential adverse effects and contraindications for the use of the sulfonamide antibiotics

Answer 22

• Acute hemolytic anaemia in humans not seen in dogs
• Keratoconjunctivitis sicca
  
  • Relatively common with acute or chronic therapy
• Reversible substrate inhibitor of thyroid peroxidase
  
  • Reversible hypothyroidism can occur with high doses
• Immune stimulating properties are due to a deficient acetylation pathway
  
  • This leads to oxidative metabolism of the drug, the metabolites of which are immunogenic
  • This can lead to immune-complex mediated hypersensitivity disorders
• Cats may develop azotemia and renal failure

Question 23

List the commonly used quinolone antibiotics in small animal veterinary medicine

Answer 23

• Enrofloxacin
• Marbofloxacin
• Orbifloxacin
• Difloxacin
  
  • All second generation
• Pradofloxacin
  
  • Third generation

Question 24

Describe the mechanism of action of the second generation fluoroquinolones

Answer 24

• The quinolones prevent bacterial DNA synthesis by inhibition of the bacterial topoisomerase
  
  • TP-II ⇒ DNA gyrase
• Limited plasma protein binding, lipophilic with high bioavailability and wide tissue distribution
• Concentration dependent killing effect
  
  • Higher single dose cf MIC is more important that persistantly elevated plasma levels
• The drug concentrate in phagocytic cells - up to 100 times that of the plasma
• Variable extent and rate of biotransformation, elimination rate and route of excretion
  
  • eg. enrofloxacin is converted to ciprofloxacin
    
    • 50% is excreted in urine as ciprofloxacin and its metabolites
  • eg. 50% of marbofloxacin is excreted unchanged in the urine, 50% excreted unchanged in the bile
• Food and antacids decrease GI absorption

Question 25

Discuss the potential clinical indications for the use of the quinolone antibiotics in dogs and cats

Answer 25

• Quinolones are primarily active against gram negative and some facultative anaerobic bacteria
• Many gram positive bacteria are susceptible, though the MIC is greater than for gram negative bacteria
• Some activity against some Rickettsiae, Chlamydiae, Mycoplasma, Bartonella and some mycobacteria
• The primary indication is for treatment of recurrent urinary tract infections and bacterial prostatitis
• Use in conjunction with doxycycline is preferred for intracellular organisms such as Brucella to limit the chance of relapse

Question 26

Describe the mechanism of resistance development to quinolone antibiotics.

Answer 26

• Resistance to the quinolone antibiotics is chromosomally mediated
• Cross resistance between the various quinolones is possible
• Development of point mutations in the topoisomerase enzyme can induce resistance
• Induction of other enzymes
• Development of a drug-efflux pump
  
  • This can contribute to multi-drug resistance

Question 27

How should quinolones be used to minimise the risk of developing bacterial resistance?

Answer 27

• Only use the particular antibiotic when there is known or likely sensitivity
• Other antibiotics should be used first when appropriate
  
  • Eg amoxycillin/clavulanate for basic urinary tract infection
• Avoid the use of doses that are minimally above the MIC
  
  • Low drug concentration provides selection pressure for resistant strains
• The dose should be above the mutant prevention concentration (MPC).
• Restricted use in food-producing animals - limit development of resistance in zoonotic bacteria
• Resistance is especially a problem with P. aeruginosa and Enterococcus sp.

Question 28

Describe the potential adverse effects and toxicities that have been described with quinolones in both dogs and cats

Answer 28

1. Gastrointestinal tract
   
   • mild anorexia, vomiting, diarrhoea
2. Neurological
   
   • CNS
     
     • Inhibition of GABA and stimulation of NMDA receptors - primarily with large IV doses
   • Eye - cats
     
     • Retinotoxicity and photoreceptor damage
     • Dose related toxicity
3. Cardiovascular
   
   • Hypotension due to histamine release with rapid IV infusion
4. Liver
   
   • Inhibit cytochrome P450-mediated drug metabolism
   • Can alter toxicity of other co-administered drugs such as itraconazole
   • May increase liver enzymes directly
5. Kidney
   
   • Crystalluria can be seen primarily with high doses
6. Skeleton
   
   • Inhibit proteoglycan synthesis leading to cartilaginous damage, especially in growing dogs
     
     • As they chelate magnesium, this problem can be exacerbated by hypomagnesemia
7. Skin
   
   • High affinity for melanin
   • Combination with melanin and UV light results in cytotoxic photodegradation products and inflammation
8. Reproductive organs
   
   • DNA inhibition effects in the developing foetus
   • Impaired spermatogenesis and testicular atrophy with chronic use

Question 29

Discuss the aetiology of Hemotropic mycoplasmosis

Answer 29

• Haemotropic mycoplasma are wall-less bacteria that attach and grow on the surface of red blood cells
• Gram-negative and non-acid fast
• They contain both DNA and RNA and replicate by binary fission
• They can be rod-shaped, sperical or ring shaped and found individually or in chains on the red blood cells
  
  • The organisms live epiceullularly
• 3 species known to infect cats
  
  • M. haemofelis
  • Candidatus mycoplasma haemominutum
  • Candidatus mycoplasma turicensis

Question 30

Discuss the prevalence of feline infection with Mycoplasma hemofelis and the other haemotropic mycoplasma organisms

Answer 30

• Prevalence varies depending on the geographic location and the study population
  
  • When anaemic cats were assessed, 30% were suspected as having hemotropic mycoplasmosis in a USA study
  • In Australia ~ 0.9% of a flea-infested population of cats were PCR positive for M. haemofelis, and 15.3% positive for Candidatus M. haemominutum
• Studies looking at clinically affected cats show that M. haemofelis is the predominant cause of anaemia and illness

Question 31

Describe the transmission of Mycopasma hemofelis infection in cats

Answer 31

• Transmission is likely via dissemination of infected blood via blood-sucking arthropods such as fleas, mosquitos and ticks
• In utero transmission may occur but the significance is unknown
• Transmission in fighting males is likely

Question 32

Discuss the phases of infection with Mycoplasma hemofelis infection in cats

Answer 32

• Disease severity can vary from life-threatening to non-clinical

4 phases of infection are recognised:

• Preparastiemic
  
  • 1-3 weeks after initial innoculation
• Acute
  
  • Time from initial parasitemic episode to the last
  • Generally lasts ~ 1 month or more
  • Organism numbers increase of 1-5 days and then disappear from the blood film - likely due to splenic sequestration and removal
• Recovery
  
  • Time from last parasitemic phase to stabilisation of the HCT
  • Often takes 1 month or more for stabilisation
• Carrier
  
  • Organisms appear to be harboured within pulmonary and splenic macrophages within intracytoplasmic vacuoles

Question 33

Describe the potential clinical signs seen in a cat with Mycoplasma haemofelis infection

Answer 33

• Clinical signs are dependent on the stage of disease, with severe signs possible during acute infection
• Younger cats are more likely to show clinical signs
  
  • Severe anaemia / MM palour
  • lethargy / depression / anorexia
  • Tachypnoea
  • Weight loss
  • Splenomegaly may be evident
• Icterus is only seen with rapid RBC destruction
• Rectal temperature may be increased with acute severe infection or decreased due to anaemia and shock

Question 34

Describe the common laboratory abnormalities seen in cats with clinical M**ycoplasma haemofelis infection

Answer 34

• Anaemia
  
  • Organism may be detected on a blood smear ~ 50% of the time
  • Anaemia may be severe generally < 20%, sometimes < 10%
  • Total red cell mass may be increased when compared to HCT due to splenic sequestration
• Reticulocytosis / polychromasia
  
  • Most common and suggest a regenerative anaemia
  • Rarely acute severe hemolysis can result in a pre-regenerative anaemia (often with hyperbilirubinemia)
• Leukocyte counts are variable and platelets are usually normal
• Autoagglutination and Coombs tests are often positive
• Erythroid hyperplasia in the bone marrow with an increase in immature stages is common
  
  • May see erythrophagocytosis in peripheral blood or bone marrow
• Plasma protein may be normal or mildly increase
• ALT/AST may be mildly increased
  
  • likely due to hepatic hypoxia associated with anaemia
• Mild increases in urea are possible and mostly caused by dehydration (pre-renal)

Question 35

Discuss the testing options that can confirm a suspected diagnosis of Mycoplasma haemofelis

Answer 35

• Close examination of a well-prepared thin blood smear using fresh whole blood can identify the organism
  
  • Wright-Giemsa stain makes identification easier than Diff-Quik
  • Exam prior to therapy and tetracyclines will rapidly remove organisms
  • EDTA storage can cause organism detachment
• Regenerative anaemia with a positive Coombs test, autoagglutination or erythrophagocytosis is suggestive of the diagnosis.
  
  • Need to exclude primary and other secondary causes for IMHA
• Real-time PCR is both sensitive and able to semi-quantify the hemotropic mycoplasmas
• Consider testing for other diseases including FeLV and FIV, especially if the anemia is not or does not become regenerative

Question 36

Discuss the treatment options for cats with Mycoplasma haemofelis infection.

What is the typical outcome following infection?

Answer 36

• Supportive therapy is recommended as required and may include:
  
  • IV fluids
  • Blood transfusion
  • Glucocorticoids for severe anaemia to minimise erythrophagocytosis
    
    • Generally not needed/required
• The first-line antibacterial drug of choice is doxycycline
  
  • Initial response can be rapid
  • ~ 2-4 weeks of treatment is generally recommended
  • PCR following treatment has been recommended
  • Follow up with 2 weeks of marbofloxacin has been recommended for severe infection or relapsing infection
    
    • May help to clear the infection completely

Most cats can recover with rapid diagnosis and treatment. Chronic carrier status is the most common outcome following acute infection. Relapses are rare but immunosuppression and other disease may precipitate relapse or positive PCR test results