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Lecture 24

Antimicrobials 3 – antimicrobials affecting DNA synthesis; antimicrobials

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affecting DNA synthesis; antimicrobials affecting metabolic pathways; assays

of antimicrobial activity

Objectives• Describe the different ways that antibiotics can inhibit bacterial DNA

synthesis and give specific examples• Describe the action of rifamycins• Describe the action of rifamycins• Describe the mode of action of novobiocin• Describe the mode of action of quinolones• Describe the mode of action of nitroimidazoles and nitrofurans and

give some examples• Describe the mode of action of sulphonamides and trimethoprim –

and the synergistic action when they are used together• Describe the methods for testing bacteria for antibiotic sensitivity –

disc diffusion MIC; MBC; Define MIC and MBC

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disc diffusion, MIC; MBC; Define MIC and MBC• Describe the relationship between MIC and disc diffusion zone

diameters• Discuss the situations in which microbial assays are conducted –

therapeutic monitoring; pharmacokinetics; bacteridical kinetics; antibiotic residues.

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Inhibitors of nucleic acid synthesis

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Rifamycins• Produced by Amycolatopsis

mediterranei• Rifampin is the most important

antibiotic – other in class include rifamycin, rifampicin, rifabutin

• Bactericidal • Mostly used in TB treatment• Mode of action – inhibits DNA

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Mode of action inhibits DNA dependent RNA polymerase –binds to the β-subunit of the enzyme and causes abortive initiation of RNA synthesis –no mRNA is produced

rifampicin

Novobiocin• Produced by

Streptomyces spp• novobiocin

Streptomyces spp• Coumarin antibiotic• Inactivates the β-subunit

of DNA gyrase – inhibits supercoiling

• Usually bacteriostatic

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• Narrow spectrum –mostly used topically against S aureus

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Quinolones• Synthetic chemical compounds• Bactericidal • Inhibit the activity of DNA gyrase which is responsible for the coiling of the• Inhibit the activity of DNA gyrase which is responsible for the coiling of the

bacterial chromosome. – Action is specific to this enzyme – so selective toxicity is good.

• Naladixic acid is the original antibiotic in this class – rarely used now. • Newer compounds in the class have a fluorine residue – so are called

fluoroquinolones – introduced in 1980s . – Norfloxacin used to treat urinary and gastrointestinal tract infections; – Ciprofloxacin – Gram-negative bacteria; – Moxifloxacin and Gatifloxacin – extended spectrum so also effective

against Gram positive organisms eg those causing pneumonia

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against Gram-positive organisms eg those causing pneumonia. • Can be administered orally. • Side effects can be a problem. • Damage joints in immature animals – so should not be used in children or

pregnant women

Inhibition of DNA Gyrase

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Quinolones

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5-Nitroimidazoles• Metronidazole (Flagyl) and

tinidazole are examples • Wide spectrum of activity

against obligate anaerobes and intestinal protozoa

• Cause extensive breakage of DNA strands and inhibition of DNA repair

• Side effects frequent – no alcohol

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alcohol

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Nitrofurans• Largely banned now

because of mutagenic tiproperties

– Damage to DNA (similar action to 5-nitroimidazoles)

• Some still used topically• Mode of action

– Prevent mRNA translation– DNA??

• Products include

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• Products include– furazolidone– nitrofurantoin– nitrofurazone

Sulphonamides• Synthetic chemical compounds – structural analogues of para-amino

benzoic acid (PABA).• Sometimes considered as metabolic pathway inhibitors and• Sometimes considered as metabolic pathway inhibitors and

sometimes as nucleic acid synthesis inhibitors • Compete with PABA in the folic acid synthesis pathway.• Pathway produces purines and pyrimidines – building blocks of

nucleic acids. • Selective toxicity is on the basis that bacteria synthesise their folic

acid but humans cannot synthesis folic acid (it must be provided in our diet).

• Sulphonamides are broad spectrum bacteriostatic agents effective

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Sulphonamides are broad spectrum bacteriostatic agents effective against Gram-positive & Gram-negative bacteria.

• Can be used orally. • Used to be used extensively for urinary tract infections – but have

significant side effects – dermatitis and rarely bone marrow suppression

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Comparison of the structure of sulphonamides and p-aminobenzoic acid

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Trimethoprim • A diaminopyrimidine – pyrimidine

analogue• Inhibits bacterial dihydrofolateInhibits bacterial dihydrofolate

reductase by combining with the enzyme

• Enzyme is present in human cells but trimethoprim has much greater affinity for the bacterial enzyme

– Selective toxicity based on differential affinity for DHF from different sources (eg mammalian vs bacterial)

• Can cause nausea• Trimethoprim-sulphonamide

combination has broad range of

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• Displays synergism when used in combination with sulphonamides (co-trimoxazole)

• Now used on its own in paediatric preparations

activity• Still treatment of choice for some

infections – pneumocystis infections in AIDS and immunocompromised patients, Listeria monocytogenes, Salmonella typhi, Nocardia spp infection.

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Assay of antimicrobial agents

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Assays1. Antibiotic sensitivity tests (AST)• Determine the sensitivity of organisms to• Determine the sensitivity of organisms to

antimicrobial agent– Isolates from an individual patient– Surveillance and monitoring

2. Assay of antimicrobial agents• Determine the concentration of antibiotic

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– Patients plasma or urine– Pharmacokinetic studies in animals (pre-

registration)

Antimicrobial Sensitivity Testing (AST)-Methods

1. Agar gel diffusion – Kirby Bauer methodL lt f b t i• Lawn culture of bacteria

• Place discs containing specified concentration of different antibiotics on the plate

• Incubate and measure the diameter of the zone of inhibition of growth around the disc

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• Use tables to assess if zone size indicates resistance or sensitivity to that antibiotic

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Kirby Bauer disc method

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Interpretation of zone sizes Antibiotic disk resistant intermediate sensitive

Ampicillin 10 μg < 12 12 - 13 > 13

Chloramphenicol 30 μg < 13 13 - 17 > 13Erythromycin 15 μg < 14 14 - 17 > 17

Gentamicin 10 μg ≤ 12.5 > 12.5

Streptomycin 10 μg < 12 12 - 14 > 14

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p y μg

Tetracycline 30 μg < 15 15 - 18 > 18

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AST - methods

2. E-testS i i i di f– Strips containing a gradient of antibiotic are placed on lawn of bacteria and incubated overnight. Minimal Inhibitory Concentration (MIC) is determined at point where zone of inhibition intersects scale on strip

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strip.– Combines ease of Kirby Bauer with

an MIC method. Particularly useful for S. pneumoniae.

http://www.uhmc.sunysb.edu/microbiology/7

AST - methods3. Agar or broth dilution – determination of Minimal

Inhibitory Concentrations (MICs)y ( )• Make up series of agar plates or nutrient broth

media containing a dilution series of the antibiotics of interest

• Inoculate with the test organism• Incubate and examine for growth

– lowest concentration of no visible growth = Minimal

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lowest concentration of no visible growth Minimal Inhibitory Concentration

– Lowest concentration of subcultured broth with no growth on a plate = Minimum Bactericidal Concentration

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Broth dilution MIC

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MIC

*Minimal inhibitory concentration

SIG

BIB

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MBC

http://pharmacology.medicine.pitt.edu/content/12

Minimum bactericidal concentrationMinimum concentration of antibiotic required to kill the organism

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Minimal Inhibitory Concentrations and Minimum bactericidal concentrations

• MICs can also be estimated from disk diff i ltdiffusion results

• Tables constructed from studies comparing agar or broth MIC results with disc diffusion zone diameters

• MBCs – need to subculture from broth

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• MBCs – need to subculture from broth cultures showing no growth

Determine correlation between disc diffusion zone diameter & broth (or agar) MIC

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Comparison of Kirby Bauer and MIC result

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Defining what is sensitive and what is resistant

Biological breakpoints

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Definition of resistance: Biological

Based on biological breakpoints an organism is considered resistantif the MIC or zone of inhibition is greater than the normal distribution ofMICs or zones for isolates lacking a specific resistance mechanism.

MIC MIC

Ideal distribution Non-ideal distribution

(a) (b)

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Susceptible Resistant low intermediate high

Biological breakpoints are easy to define if the MIC distribution is bimodal (a) or polymodal, less so, if the distribution is continuous (b).

intermediate

Importance of MIC

• Allows comparison with pharmacokinetic d t t if hi bl l l l illdata to see if achievable plasma levels will be adequate to control/kill the organism

• Establish relationship between biological sensitivity/resistance and clinical sensitivity/resistance – not always the

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sensitivity/resistance not always the same!

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Standardisation of AST

• Critically importantN b f id li CLSI (Cli i l &• Numbers of guidelines eg CLSI (Clinical & Laboratory Standards Institute)– increasingly being adopted world-wide– standardise the way the testing is carried out– Standardise interpretation

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– Quality control of media, antibiotic disks or solutions

– Quality control organisms

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Antibiotic assays

• Therapeutic monitoring• Determining potency• Studying pharmacokinetics• Assessing antimicrobial activity of new

products

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• Detection of residues in foods, environmental samples

Metabolism of Antibiotics

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CHARACTERISTICS OF THE AMINOGLYCOSIDES

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Therapeutic drug monitoring is essential

http://pharmacology.medicine.pitt.edu/content/10

Peak

AMINOGLYCOSIDE TOXICITY

Toxic level

µg/mL

Peak

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Trough

http://pharmacology.medicine.pitt.edu/content/11

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Bactericidal kinetics

• Kill curves of oral microorganismsmicroorganisms exposed to a particular mouth wash

38http://www.inet.bg/~usmeds/surechoice/research/mouthwashres2.html

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Antibiotic residues

• Residues can be found – meat or milk if animals have been treated with

antibiotics too close to slaughter– Sewage

• Doubtful public health concern – except for allergies (eg penicillin)

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g ( g p )• Trade issue (animal products)• Environmental concerns

Antibiotic residues - methods• Microbiological screening

– Testing urine, meat juice, sewage for presence ofsewage for presence of substances that inhibit the growth of particular bacteria known to be sensitive to particular antibiotics

• HPLC – detection of specific compounds (confirmatory test for microbiological screening)

• Enzymatic assay – eg detection of β l t i ilk

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β-lactams in milk– hydrolysis of a 3-peptide (Ac-

L-Lys-D-Ala-D-Ala) into a 2-peptide (Ac-L-Lys-D-Ala), a reaction which is inhibited in the presence of beta-lactams

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Australian National Residue Survey 2005-2006

Species Number of samples

Number of tests

Residues in edible tissues

cattle 5,543 78,384 2 (sulphonamide, neomycin)

sheep 4,692 58,253 0

pig 2,871 26,920 0

poultry 334 9,411 0goat 249 3,819 3 (moxidectin)

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g , ( )

horse 141 2,189 0

Other meat species

442 8,971 0

eggs 75 1,000 2 (lasolocid, nicarbazin)

http://www.daff.gov.au/__data/assets/pdf_file/0009/182556/results_report_0506.pdf