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MCB 3020, Spring 2005

Microbial Growth Control and Antibiotic Resistance

2Chapter 18 Microbial Growth Control:I. Microbial growth controlII. Measuring antimicrobial activityIII. Food preservationIV. Antimicrobial drugsV. Antibiotic Resistance

3I. Microbial Growth Control

A. usesB. autoclaveC. radiationD. filtersE. chemical agents

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4I. Microbial Growth Control

SterilizationKilling or removal of all living organisms and their viruses

Inhibition limiting microbial growth

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5A. Uses

Food preservationLaboratory workDisease preventionDisease treatment

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6B. AutoclaveMachine that usessteam under pressure for sterilization.

Items are heated to121°C for 10-15 minutes.

kills endospores

Autoclave

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7C. Radiation1. Ultraviolet (220 to 300 nm)

poor penetrating powerused to disinfect surfaces and air

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2. Gamma and X-rays

good penetration

used for food preservation and sterilization of surgical supplies

ionizing radiation

8D. Filters

Used to sterilize heat-sensitive solutions and gasses

A pore size of 0.22 micron will remove most bacteria.

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Will it remove most viruses?

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Antimicrobial agentsChemicals that kill or inhibit the growth of microorganisms

Cidal agentsChemicals that kill(bacteriocidal, fungicidal, viricidal)

E. Chemical Agents

10Bacteriostatic agents

chemicals that inhibit growth, but do not killfrequently are inhibitors of protein synthesis

Bacteriolytic agentskill cells by lysiseg. penicillin

111. DisinfectantsChemicals used to kill microbes oninanimate objects.

ChlorinePhenolic compounds

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122. AntisepticsChemicals used to kill microbes onliving tissue.

Alcohol (70% on skin)Hydrogen peroxide

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13II. Measuring antimicrobial activity

A.Tube dilution assayB. Agar diffusion assay

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14A. Tube dilution assay

1. inoculate tubes containing several concentrations of test compound with test organism and incubate.

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1 .1 .01 .00110

MIC

bacterialgrowth

MIC (minimum inhibitory concentration)The lowest concentration of a substance that inhibits growth of a test organism TB

16B. Agar diffusion assay

agar plate

lawn of test bacteria

filter paper soakedwith test compound

zones of inhibition(no growth)

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17III. Food preservationA. Common spoilage organismsB. Preservation methods

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18A. Common food spoilage organismsMeat

enteric bacteriaEscherichia coliSalmonella

Milk productslactic acid bacteria

Fruits and vegetablesErwiniaPseudomonas TB

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1. PasteurizationHeat treatment to reduce the number of viable organisms.71°C, 15 sec., or 63-66°C, 30 min

milk juice wine etc.

Used on foods the would be ruined by higher temperatures.

B. Preservation methods

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202. Temperature control

refrigeration and freezing

3. Sterilizationcanning

4. Controlling water availabilityadding salt (eg. ham)adding sugar (eg. jelly)

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215. pH control

picklingfermented foods

6. Chemical preservativesNa propionateNa benzoate

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22IV. Antimicrobial drugs

B. Growth factor analogsC. AntibioticsD. Antivirals and antifungals

A. Selective toxicity

23A. Selective toxicity

Toxicity for the pathogen, but not for the host.

Something to think about:What is the basis for selectivetoxicity?

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24B. Growth factor analogsA substance structurallyrelated to a growth factor that blocks its use.

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251. Sulfanilamide

Growth factor analog structurallyrelated to p-aminobenzoic acid (PABA)

Inhibits microbial growth byinhibiting folate synthesis

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H2N SO2NH2

sulfanilamide

H2N COOH

PABA

NHN

H2NN

N CH2 NH

C

O

Rfolate

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Sulfanilamide is nontoxicto humans because we take up folate from our diet.

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28C. Antibiotics

Substances produced by microbesthat kill or inhibit the growth of microbes

bacteriostatic agents inhibit growth

bacteriocidal agents kill

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291. inhibitors of cell wall synthesispenicillinvancomycin

2. inhibitors protein synthesiserythromycin (50S ribosomal subunit)

tetracycline (30S ribosomal subunit)

streptomycin (30S ribosomal subunit)

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HN

ON

SCH3

CH3H

COO-H

beta-lactam ring

R

natural penicillin R = CH2-CO-

Prevents transpeptidation in cell wall

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CH3

CH3

CH3

CH3

CH3

OCH3

H3C

H3CH2C

H2C OH

O

H3C

O

HOO

CH3

HO

O

NCH3H3C

O

HO

OH

O

O

Erythromycin (macrolide)

macrolide ring 50S ribosomal subunit

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R4 R2 R3 NCH3H3C

R1 H

OH O OHOH

O

OH

CO

NH2

Tetracycline: R1=H, R2=OH, R3=CH3, R4=H

• Broad spectrum• Target: 30s ribosomal subunit

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H2C-NH2

O

OH

OHHO O

NH2

O

OH

O

NH2

CH2OHO

NH2

OHHO

Kanamycin (aminoglycoside)

Target:30 s ribosomal

subunit

343. inhibitors of DNA gyrase

naladixic acidnovobiocin

4. inhibitors of RNA synthesis

rifampin

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35Antivirals and Antifungals

Chemicalsrifampinazidothymidine (AZT)

Interferoninhibits viral RNA synthesis

A. Antivirals

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36B. AntifungalsErgosterol inhibitors

polyenesazoles

Selective toxicity is more difficult toobtain with antivirals and antifungals

Important point

Why? TB

37V. Antibiotic Resistance

A. the problem of resistanceB. resistance mechanismsC. development of resistanceD. enzymes that inactivate antibiotics

38A. The problem of resistance

• Vancomycin-resistant Staphlyococcus aureus

• Penicillin-resistant Streptococcus pneumoniae

• Quinolone-resistant Salmonella enterica

Examples of drug-resistant bacteria

39Why so much resistance?

• Overuse of antibiotics in inpatient and outpatient settings.

• Increased use of quinolones, tetracyclines, and glycopeptides in agriculture, the poultry industry, veterinary practice, and marine biology.

• Newer, implantable cardiovascular and orthopedic devices that necessitate prophylactic antibiotics.

40B. Resistance mechanisms

• lack of target site• impermeability• chemical modification of the antibiotic

• pump antibiotic out of cell

41C. Development of resistance

1. Mutationtarget site modification

2. Gene transferR-plasmids

42R-plasmids (resistance plasmids)Plasmids that carry antibiotic resistance genes.

Antibiotic resistance genes usuallyencode enzymes thatinactivate antibiotics

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3. Tetracycline pumppumps tetracycline out of the cell

D. Enzymes that inactivate antibiotics

1. Chloramphenicol acetyltransferaseacetylates chloramphenicol

beta-lactamasecleaves the beta-lactam ring

44Interactions of antibiotics with alcohol in humans

• Antibiotics that are affected by alcohol are chloramphenicol, cephalosporins, metronidazole, and others.

• These produce "disulfiram-like" reactions.

45Disulfiram-like reactions

• Disulfiram is a drug to treat alcoholism.• Some antibiotics cause a reaction similar to disulfiram

reactions.• Inactivates the enzyme aldehyde dehydrogenase.• Causing accumulation of acetaldehyde in blood.• Symptoms are flushed face, severe headaches, chest

pains, shortness of breath, vomiting, and sweating.

46Ethanol

NAD+

NADH

NAD+

NADH

Acetaldehyde

Acetate

Acetyl CoA

Alcohol dehydrogenase

Aldehyde dehydrogenaseX

Disulfiram

47Study objectives1. Know how the following are used to control microbial growth: autoclaves, radiation, filters, disinfectants, antiseptics. Contrast cidal agents, bacteriostatic agents,and lytic agents. Know the examples presented in class. 2. Compare and contrast the tube dilution assay and the agar diffusion assay. Understand how each is used to measure antimicrobial activity of chemicals.3. What is MIC? How does it correlate with antimicrobial activity of an inhibitor?4. Memorize the common food spoilage organisms covered in class. 5. Know what pasteurization is and what types of foods are pasteurized.6. Memorize the food preservation methods and examples presented in class. 7. What is selective toxicity? Understand the basis of selective toxicity of growth factor analogs, antibiotics, azidothymidine, interferon, and antiviral agents.8. What is a growth factor analog?9. How does sulfanilamide inhibit the growth of some bacteria?10. Know the names and targets of the antibiotics presented in lecture.11. Know the following antiviral agents:rifampin, azidothymidine, interferon. Azidothymidine inhibits reverse transcriptase. What viruses are affected this?12. Know the names and target of the antifungal agents (polyenes, azoles).13. Why is selective toxicity more difficult to obtain with antivirals and antifungals?

48Study objectives14. Give 3 examples of antibiotic-resistant bacteria. What are the major causes of antibiotic resistance? 15. What are the mechanisms by which bacteria require antibiotic resistance? What is the role of R-plasmids in resistance? Understand how antibiotic-inactivating enzymes work. 16. Explain why alcohol should not be consumed while taking some antibiotics. Why do disulfiram-like antibiotics cause symptoms when consumed with alcohol?