1. Antibacterials Review
November 6, 2009

2. Cell-wall active agents
Penicillins
Narrow spectrum:
penicillinase susceptible
penicillinase resistant
Broader spectrum
Cephalosporins
Narrow spectrum (1° generation)
Broader spectrum (2-4° generations)
Carbapenems
Aztreonam
Vancomycin

3. Penicillins
All derived from 6-aminopenicillanic acid and have b-lactam ring
Vary in resistance to stomach acid, polar, not metabolized easily
usually excreted unchanged in urine both via glomerular filtration and tubular secretion (latter is inhibited by Probenicid)
Ampicillin and nafcillin also excreted in bile
Procaine/benzathine forms of penicillin G injected i.m. to give long half-life, otherwise plasma half life is short (30-60 min)
Most only cross blood brain barrier when meninges are inflamed.

4. Penicillins Bactericidal Targets (peptidoglycan synthesis)
Penicillin binding proteins (PBPs)
Transpeptidase
Activate some autolytic enzymes
Resistance usually due to b-lactamases
(esp. Staph and many Gram-negatives)
Resistance also known via changing PBPs
(methicillin-resistance in Staph
(PenG resistance in pneumococcus)
Porins mutations in Pseudomonas may allow resistance

5. Clinical use of penicillins
Narrow spectrum, penicillinase-susceptible drugs
penicillin G (parenteral – unstable in stomach acid)
penicillin V (given orally)
Used for streptococci, meningococcus, Gram-positive bacilli (Bacillus), Spirochetes (Treponema)
Resistance reported for
most Staph aureus,
many Neisseria gonorrhoeae
many Strep pneumoniae

6. Clinical use of penicillins (cont.)
Very narrow spectrum, penicillinase-resistant agents
(methicillin)
nafcillin
oxacillin
Primarily used for infections by Staph. aureus and Staph. epidermidis
Resistance seen with MRSA and with some organisms making ESBLs

7. Clinical use of penicillins (cont.)
Broader spectrum, penicillinase-susceptible agents
Ampicillin and amoxicillin
Used for PenG susceptible agents
and for
enterococci, Listeria, E. coli, Proteus, Haemophilus influenzae, Moraxella catarrhalis
Used in combination with penicillinase inhibitors (e.g. clavulanic acid)
Synergy shown with aminoglycosides for Listeria and enterococci

8. Clinical use of penicillins (cont.)
Broader spectrum, penicillinase-susceptible agents (cont.)
Piperacillin and ticarcillin
Active against several Gram-negative rods
Pseudomonas, Enterobacter, some Klebsiella
Synergistic with main glycosides
Often used with penicillinase inhibitors

9. Toxicity of penicillins
Nausea, diarrhea are fairly common, especially with oral penicillins
Allergy (including anaphylaxis or induction of Type II and III reactions). Persons with allergy may be desensitized if necessary using a ~3-4 h rapid desensitization protocol
Methicillin may cause nephritis
Nafcillin may cause neutropenia
Ampicillin often causes a maculopapular rash. This can be very pronounced if ampicillin is given to someone with mononucleosis (EBV) when this reaction is almost diagnostic for infective mononucleosis.

10. Cephalosporins Most administered parenterally
Many partly metabolized by liver but still usually excreted in urine (like penicillins)
But cefoperazone & ceftriaxone (mainly via bile)
Most 1° and 2 ° generation do not enter CSF (even if meninges inflamed)
Targets same as penicillins – i.e, bactericidal drugs
Tendency to be more resistant than penicillins to b-lactamases, but not to all.
Note: MRSA is resistant to cephalosporins

11. 1st generation cephalosporins
Cefazolin (parenteral) and cephalexin (oral)
Used for Gram-positive cocci
Many E. coli and Klebsiella pneumoniae
NOT for
Gram-neg cocci
enterococci
MRSA,
most Gram-neg rods

12. Second generation cephalosporins
Extended coverage of Gram-negatives
Less coverage of Gram-positives
Diverse activity shown by different members
Cefotetan, cefotoxin (Bacteroides fragilis - anaerobe)
often resistant to ESBLs
Cefomandole, cefuroxime, cefaclor
(H. influenzae. M. cattharalis)

13. Third generation cephalosporins
Extended coverage of Gram-negatives
Enter CSF (except cefoperazone and cefiximine)
Resistance associated with extended spectrum beta-lactamases (ESBLs in Klebsiella and E. coli especially)
Active against several Gram-neg including Neisseria
ceftriaxone parenteral and cefiximine oral for gonococcus)
cefoperazone & ceftazidime active against Pseudomonas
One injection of ceftriaxone (~ 8h half life) usually effective for acute otitis media

14. Fourth generation cephalosporins
Cefepime
Expands coverage of 3rd generation to include activity against Gram+ found in 1st generation cephalosporins
Used for penicillin-resistant pneumococci and for several b-lactamase producing Gram-negatives including Enterobacter

15. Cephalosporins - Toxicity
Allergy (cross-reactivity within cephalosporins as group)
Some cross-reaction with penicillins
Pain at injection site
Phlebitis after i.v. administration
May increase nephrotoxicity of aminoglycosides
Disulfuram reaction with some (e.g. cefoperaxzone, cefotetan, cefamandole)

16. Other b-lactams Aztreonam (a monobactam. Acts on PBP3)
No activity on Gram-positive bacteria or anaerobes
Resistant to b-lactamases produced by many Gram-negative rods including Klebsiella and Pseudomonas but not resistant to ESBLs.
Synergistic with aminoglycosides
Prolonged half-life in renal failure
No significant cross-allerginicity with penicillin

17. Other b-lactams Imipenem, meropenem (carbapenems)
Wide activity against Gram positive cocci, Gram-negative rods, and anaerobes
Used with aminoglycoside for Pseudomonas infection
Resistant to ESBLs
Imipenem is administered with cilastin to prevent inactivation by renal dehydropeptidase
CNS toxicity at high concn

18. Vancomycin Bactericidal glycoprotein inhibiting cell wall formation
Binds D-Ala-D-Ala pentapeptide side chain to prevent transpeptidation in growing peptidoglycan
Resistance in VRE and VRSA is due to change of one D-ala to D-lactate
Narrow spectrum of use
Mainly for MRSA, penicillin-resistant pneumococcus and C. difficile
Eliminated unchanged in urine. Not absorbed from gut so only used orally for C. difficile
Nephrotoxic, ototoxic, RED MAN syndrome with rapid infusion

19. Tetracyclines: Mode of Action
Reversible binding to 30S ribosome subunit blocking aminoacyl-tRNA access to acceptor site (A site) X

20. Tetracyclines
Drugs usually given orally and absorbed from small intestine
BUT Interference of uptake by food, divalent and trivalent cations (Ca++, Mg++, Fe++, Bi +++, Al+++ ) in antacids, dairy products.
Active against many Gram + and Gram – bacteria including anaerobes, rickettsiae, chlamydiae, mycoplasmae.
Tigecycline has wider range.
Must be given i.v.
MRSA included, but NOT Pseudomonas nor Proteus
Some tetracyclines act against protozoa and those filarial nematodes that have endosymbiotic bacteria
(e.g. doxycyline: Entamoeba and Plasmodium falciparum)

21. Tetracyclines Enter all body fluids well except CSF
(~10-20% plasma level)
Actively excreted in bile and into feces
some enterohepatic circulation
Also excreted in urine
But NOT doxycycline nor tigecycline

22. Tetracyclines toxicity
Allergies
Minor effects on liver
Vestibular toxicity
Occasional photosensitivity
Kidney excretion means doses need to be watched in renal failure
Special effect on growing bones
Enter breast milk and cross placenta
Chelation with calcium causes binding to growing bones/teeth - damage

23. Tigecycline (Tygacil)
Became available 2005
Has very broad spectrum of activity including
MRSA, VRSA, VISA, and coagulase-neg Staph
Penicillin resistant and susceptible streptococci
Enterococci (including VRE)
Gram-positive rods
Enterobacteriaceae
Acinetobacter (multidrug resistant)
Anaerobes (Gram + and -)
Chlamydiae, rickettsiae, Legionella,
fast growing mycobacteria
NOT effective against Pseudomonas or Proteus where the efflux pump is effective at removing it thereby causing intrinsic resistance.

24. Macrolides – block transpeptidylation

25. Macrolides Erythromycin, azithromycin, clarithromycin
Oral bioavailability
Active against Campylobacter, Mycoplasma, Legionella, Gram-pos cocci, some Gram-negs
Erythromycin widely used for community acquired pneumonia

26. Macrolides Azithromycin Clarithromycin
Concn in tissues and phagocytes higher than plasma. Slow release allows once a week dosing (half life 2-4 days). Especially useful for chlamydial infections
Clarithromycin
Used for Mycobacterium avium

27. Aminoglycosides Broad spectrum Gentamicin Amikacin Tobramycin
Netilmicin
Those with limited clinical application
Streptomycin
Neomycin
Kanamycin
Do not work alone on serious infections by enterococci or
streptococci but can increase antimicrobial activity of other
drugs when used in combination for these infections

28. Mistranslation – one effect of aminoglycosides
Gentamicin in major groove of RNA
at site where aa-tRNA interacts with mRNA. Distortion of ribosomal site by antibiotic causes misreading of codons.
Interaction is within the 30S subunit
puglisi.stanford.edu/research.html

29. Aminoglycosides Highly charged (polycations) and poor lipid solubility
Do not penetrate human cell cytoplasm well.
Generally given by i.v. or i.m. injection.
Mainly excreted in urine
Action independent of microbial concentration so very useful for intrabdominal infections
Persistent suppression of microbial growth after dropping to non-lethal level (post antibiotic effect)
High dose once a day is more effective and less toxic than same amount split and administered in 3 doses per day
Toxicity largely depends on time the drug remains above a toxic concentration

30. Aminoglycosides - Toxicity
Neuromuscular blockade
binding calcium in presynaptic region – reversible with calcium gluconate
Nephrotoxicity
interference with tubular function including excess loss of Mg and Ca: increased toxicity in combination with vancomycin, amphotericin B, diuretics, several others. Generally reversible
Ototoxicity (auditory and vestibular)
non-reversible

31. Novel uses in the works for aminoglycosides
Treatment of genetic disease in humans when caused by premature stop codon in gene.

32. Spectinomycin Similar structure to aminoglycosides
Target is on 30S ribosome
Used to treat penicillin-resistant Neisseria gonorrhoeae

33. Others (all act on 50S subunit)
Chloramphenicol
Wide spectrum of activity. Significant toxicities. Inactivated by liver (liver action has low activity in newborn)
Aplastic anemia (gray baby syndrome)
Restricted uses (e.g. serious rickettsial infections)
Clindamycin (a lincomycin) MLSB resistance known
Works like macrolides
Bacteroides, several anaerobes
Increased danger of Clostridum difficile colitis
Quinupristin-dalfopristin MLSB resistance known Mixture of streptogramins
VRE if Enterococcus faecium (but not E. faecalis)
Linezolid
VRSA, MRSA,
VRE (both E. faecium and E.faecalis)
May cause mild thrombocytopenia and bone marrow suppression

34. Drugs affecting nucleic acid synthesis
Folate inhibitors
Sulfonamides (derivatives of sulfanilamide structural analog of p-aminobenzoic acid)
Trimethoprim, pyrimethamine
DNA gyrase inhibitors
Quinolones

35. Purine and pyrimidine synthesis

36. Spectrum of sulfonamides
Gram-positives and gram-negatives
Nocardia
Chlamydia
Some protozoa
Note:
Sulfonamides stimulate rickettsial growth.
Poor activity against anaerobes

37. Adverse reactions of sulfonamides
Allergies
Photosensitivity
Nausea and diarrhea
Fever and skin rashes, exfoliative dermatitis
Steven-Johnson syndrome (<1% treatment courses)
Inactivation in part in liver
Crystalluria, hematuria (drugs and inactivated forms excreted in urine and precipitate at acid pH)
Hematopoietic reactions
Hemolytic anemia or aplastic anemia
Granulocytopenia, thrombocytopenia, leukemoid
Glucose-6-DH deficiency – enhanced hemolytic reactions
If given in late pregnancy
Kernicterus (brain damage due to excess jaundice)

38. Trimethoprim (TMP) Mainly excreted in urine
Good absorption orally. Lipid solubility enhances distribution (more than sulfamethoxazole - SMX) including into CSF
Extended use gives similar side effects to sulfonamides
Reduces length of sulfonamide treatment time when used in combination with the sulfonamide
E.g. TMP-SMX for UTIs and Pneumocystis prophylaxis
Pyrimethamine-sulfadiazine for Toxoplasma

39. Quinolones Interfere with DNA gyrase and DNA topoisomerase IV

40. Activity of fluoroquinolones
Excellent activity against Gram-negatives
Lesser activity against Gram-positives but some newer agents better
(e.g. ciprofloxacin maintained for anthrax)
Bactericidal and have a post antibiotic effect

41. Uptake of Quinolones Oral agents
Uptake inhibited up to 80% by coadministration with Al and Mg-containing antacids. Some inhibition by calcium and ferrous ions.
Most also available for i.v. administration
Excreted in urine
except ciprofloxacin with 50% bile and 50% urine

42. Indications for fluoroquinolones
b-lactam-resistant gonococcus
Cystitis (where trimethoprim-sulfamethoxazole not useful)
Complicated ascending UTIs (ureter/kidney)
Prostatitis (drugs concentrate in prostatic tissue)
Single dose for gonorrhea (but resistance developing)
Pelvic inflammatory disease

43. Indications for quinolones
Respiratory tract infections
Many uses but may be need in combination with b-lactams for severe pneumococcal pneumonia or with other drugs when Pseudomonas is likely (e.g. ventilators)
Gastrointestinal infection
Very useful for Shigella (1 dose), Salmonella (including typhoid), E. coli, cholera, Campylobacter (3-5 days). Resistance a concern

44. Adverse reactions of quinolones
Nausea – vomiting - diarrhea
Some cause Q-T interval prolongation
May damage growing cartilage (not recommended in under 18 year olds) but the damage appears reversible and the drugs are likely safe for some uses
Tendinitis (rare in adults – main >50 years) Usually starts in Achilles tendon - can lead to tendon rupture
Probably should be avoided during pregnancy since safety not shown

45. Metronidazole (Flagyl®)
No net charge at physiological pH. Small molecule enters cells.
Very good bioavailability, can be given orally
Effective on most obligate anaerobic bacteria including Clostridium difficile,
and Gardnerella vaginalis but NOT useful for most Actinomyces spp.
and all Propionibacteria (e.g. P. acnes).
Effective against anaerobic protozoa (Trichomonas, Giardia, Entamoeba)
Activated after being reduced by ferredoxin
Redox potential produced by aerobically growing bacteria not low enough
to activate the drug.

46. Ferredoxinred Ferredoxinox Metronidazole Short-lived intermediates
R-NO2 + e- → R-NO2-●
R-NO2-● + H+ → R-NO2H●
2R-NO2H● → R-NO2 + R-N(OH)2
R-N(OH)2 → R-NO + H2O
R-NO + e- → R-NO-●
R-NO-● + H+ → R-NOH●
R-NOH● + R-NO2H● → R-NHOH + R-NO2
R-NHOH + 2e- + 2H+ → R-NH2 + H2O
Free radicals (●)
fragment DNA.
The intermediates
produced by
reduction of the
–NO2 group are the
active form of
the drug

47. Antimicrobials Part II
Antimycobacterials
Detecting resistance
Probiotics

48. Treatment of mycobacterial diseases
Problems
Waxy cell walls (inhibiting drug diffusion)
Bacteria live both extracellular and intracellular
Slow growth (drugs must be used for long periods)
Many drugs only work for one or a few species
Active disease should be treated using combination therapy with multiple drugs

49. Tuberculosis
All first line agents except ethambutol are all hepatotoxic and when used together have heightened hepatotoxicity
Streptomycin (no longer first line because resistance is fairly common) is not hepatotoxic
Treatment using first line agents becomes modified based on:
Pre-existing hepatitis
Pre-existing drug resistance

50. First line agents Isoniazid (INH)
Targets cell wall fatty acid synthesis
Only for Mtb (not used for other mycobacteria)
Requires activation by Mtb catalase-peroxidase
Used alone in treatment of PPD-positive persons with latent TB infection (i.e., no active disease)
Resistance when Mtb that have lost catalase gene
Resistance shown by Mtb with altered INH targets
Liver toxicity increases with age
Other toxicities include peripheral neuropathy (often reversible by vitamin B6), lupus-like syndrome (~1% of persons though 20% develop anti-DNA)

51. Other first line agents against Mtb
Rifampin (RIF)
Good at inhibiting and killing bacteria
Used for other mycobacteria including M. leprae
Used for HIV-associated Mtb
Induces acetylating enzymes in liver thus reducing its activity as treatment continues
Hepatotoxicity, itching, and orange staining of secretions are most common side effects
Significant drug interactions (including cyclosporine, contraceptives …) since it induces many cytochrome P450 isoforms.
Can’t be used with AZT (zidovudine) since it upregulates the glucuronyl transferase that inactivates AZT.
Also used for prophylaxis of contacts of children with active H. influenzae type b disease

52. Other first line agents against Mtb
Pyrazinamide
Most hepatotoxic of first line agents
Good killing within macrophages and in caseous lesions
Not effective at alkaline or neutral pH
Deaminated in bacteria to make inhibitor of fatty acid synthase.
M. bovis is resistant (amino acid substitution in deaminase)
Polyarthraligia and hyperuricemia are main side effects
Ethambutol
Inhibits cell wall arabinogalactan formation
No liver toxicity
Significant toxicities include retrobulbar neuritis affecting visual acuity and severe skin reactions

53. Streptomycin One of numerous second line agents
Aminoglycoside works extracellularly only
Toxicities include ototoxicity, circumoral parathesias
Toxicity noted especially for Cranial nerve VIII
Not hepatotoxic

54. Other mycobacteria Dapsone
sulfonamide-like inhibits folate synthesis
Used in Multiple Drug Therapy to prevent resistance arising.
Typically dapsone is used with rifampin and clofazimine in leprosy

55. Other mycobacteria Azithromycin or clarithromycin (macrolides)
Prophylaxis for M. avium when CD4<75/ml
Azithromycin has elimination half life ~3 days (i.e. once-a week dosing is possible)

56. ANTIMICROBIAL SUSCEPTIBILITY TESTING

57. Resistance limits usefulness of antimicrobials
Need to identify isolates and also test for resistance
1. Intrinsic resistance in some species (no target)
2. Development of resistance
Blocking entry of antibiotic or upregulating export pumps
Mutation or enzymatic modification of target
Overexpressing target or upregulating alternative pathway bypassing target
Modifying antibiotic directly to inactivate it
Failing to activate antibiotic.

58. MIC – a quantitative measure of susceptibility
Minimum inhibitory concentration (MIC)
Measures concentration of drug that prevents growth in vitro (does not necessarily kill bacterium) when tested over a set period, usually 1 day.
MIC is an intrinsic property of the bacterium. It stays the same regardless of site of infection (unless strain develops resistance).
Therapy should be chosen to so the concentration of drug in the areas of infection exceeds the MIC

59. Significance of MIC for clinical practice
When the MIC for an antimicrobial is greater than safe therapeutic concentration, bacteria are resistant
When the MIC is below the safe therapeutic level, bacteria are susceptible
An increase in MIC points to resistance developing

60. MIC determination - Tube dilution assay Bacterium, growth medium and drug added to each tube incubated 24 h
Susceptible isolate (Drug only toxic above 6 mg/ml)
MIC = 1 mg/ml
Concentration of drug in tube (mg/ml)

61. Tube dilution assay - resistant isolate Isolate is resistant because drug is toxic above 6 mg/ml)
MIC = 8 mg/ml
Concentration of drug in tube (mg/ml)

62. MIC from antibiotic diffusion in agar
Inoculation of plate with clinical isolate. Antibacterial
disks are placed on surface. Plates incubated
~24 h to allow bacteria to form a lawn
Kirby-Bauer
Zone around antibiotic disc showing no bacterial growth due to presence of antibacterial diffusing out of disk. Antimicrobial is most concentrated at disk A B C

64. E-TEST® agar diffusion MIC determination
Continuous scale - not just doubling dilutions.
Expensive

65. Surrogate tests
Numerous types: tests with one drug to predict response to another
E.g. 30 mg cefoxitin disk for oxacillin-resistant Staph. aureus (i.e. MRSA)
Cefoxitin induces mecA (PBP2a) more effectively than oxacillin in those MRSA that are not constitutive producers of PBP2a

66. Macrolide - Lincosamide - Streptogramin B resistance
Staphylococcus aureus
Erythromycin R
Clindamycin S
This result could hide potential to express clindamycin resistance (MLSB pattern) due to methylase.
Erythromycin far better inducer of methylase than is clindamycin
If resistance to clindamycin induced by erythromycin (D-test), this points to MLSB resistance

67. Measuring bactericidal activity
MBC (minimum bactericidal concentration) – concentration needed to ensure all bacteria killed
Specialty test, only occasionally used when patients lack residual immunity, or when infection may require bactericidal levels such as in endocarditis/osteomyelitis

68. Extended spectrum b-lactamases ESBLs
Over 340 different b-lactamases in Gram-negative rods.
Some have very limited substrate activity
e.g. the plasmid-mediated penicillinases with little activity against cephalosporins
SHV-1- ~ 100% Klebsiella pneumoniae isolates (confers R to ampicillin+ticarcillin)
TEM-1 - ~ 50% (currently ) E. coli isolates (confers R to ampicillin).
Mutations in the genes for SHV-1 and TEM-1 → extended-spectrum b-lactamases (ESBLs)
ESBLs have activity against ALL the penicillins, most cephalosporins, and aztreonam.
Most common in Klebsiella pneumoniae, K. oxytoca, E. coli but also in other Gram-negatives

69. Many microbes exist in complex communities Therapeutic alterations to “microbiota”
Antibiotics
Chemicals directly targeting (inhibiting/killing) microbes
Prebiotics
Non–digested ingredients that selectively stimulate one or a group of bacteria in the colon: e.g. lactulose used to reduce ammonia
Probiotics
Live organisms, which when administered in adequate amounts, confer a health benefit on the host

70. Targeted antibiotics Bacteriophages
Increasing interest in bacteriophages to attack biofilms and at other sites.
Many are highly species-specific
Many bacteria attacked by a large number of different bacteriophages suggesting several targets
Rapid lysis of cell wall bright about by murein (peptidoglycan) hydrolases produced during infection (similar structures to bacterial peptidoglycan hydrolases)

71. Lysins produced during intracellular growth by bacteriophages
Lysins transferred to wall via pores
(holins) produced by phage during
infection.
Pure lysins can act at outside of
bacterial cell
Hermoso et al., Current Opinion in Microbiology 2007, 10:

72. Potential problems of bacteriophage therapy
Narrow host range of most lytic phages
Bacteria can become resistant
Antibody responses may neutralize activity
Pharmacokinetics not easy
Potential to mobilize and transfer genes
Use of phage lytic enzymes appears to avoid most of these problems. Resistance does not seem to develop

73. Probiotics
“Live organisms, which when administered in adequate amounts, confer a health benefit on the host.”
Bacteria must be able to adhere and colonize and work within context of a biofilm
Indications suggest usefulness in halitosis and maybe caries (tooth decay)
Strong evidence for value in pouchitis following ileal pouch-anal anastomosis

74. Fecal flora therapy in relapsing Clostridium difficile colitis
5% of C. difficile colitis do not respond permanently to treatment with metronidazole or vancomycin and develop a relapse
Fecal flora replacement with non-C. difficile donor has been reported effective
More research needed

75. Microbial intestinal flora may prime for allergic responses
Antibiotic-treated and Candida colonized mice
Cefoperazone
5 days
± Candida albicans orally
antigen/spore lung challenge for 2-3 weeks
Noverr et al. Infect. Immun. 2005;73:30-38

76. Candida in intestinal flora may prime for allergic responses in lung (response is IL-13 dependent)
Mice treated with cefoperazone Mice treated with cefoperazone for
for 5 days No Candida days and colonized with C. albicans
Challenge with
Aspergillus spores
4 times in 12 days
after Candida
Ovalbumin
6 times in 21 days
after Candida
increased IgE,
goblet cell metaplasia
lung eosinophils
Noverr et al. Infect. Immun. 2005;73:30-38

77. Antibiotics and asthma in humans
Antibiotic usage changes microbial flora in intestine and changes stay for significant time –
More clostridia and Candida (stimulatory lipids)
Less lactobacilli (butyrate, anti-inflammatory)
Possibly allows increased chances of developing asthma
Several papers suggest antibiotic use early in life is a risk factor for asthma
Suggestion that probiotics given infant may reduce tendency to develop asthma (some data)