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Microbial ControlTalaro Chapter 12

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Choosing a disinfectant or antiseptic….often called “germicides”

-gaseous, liquid or solid state-effective concentration (versus toxicity)-broad spectrum?-low toxicity-penetration of surfaces of inanimate objects or tissues-resistance to becoming inactivated by organic matter-noncorrosive or nonstaining properties-odor-affordability-availability

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Chemical Decontamination Procedures3 Levels

1) High - kill endospores… are sterilants – necessary for medical devices etc. e.g., catheters (some parts are not autoclavable)

2) Intermediate – Kills fungal spores but not endospores and generally kills most pathogens –used to disinfect items that touch mucus membranes but are not invasive

3) Low – Kills only vegetative cells of bacteria and fungi… probably kills most pathogens – for cleaning furniture, straps, electrodes… things that touch the skin surface

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Chemical Groups of Germicides

1) Halogens (a group of non-metallic elements– fluorine, bromine, chlorine, and iodine

Antimicrobial in the non-ionic state…Fluorine and bromine are dangerous so only Cl

and I are used routinelyBacteriocidal, bacteriostatic, and even sporicidal

with long contact timeCl and I are the active ingredients in over 1/3 of

all antimicrobials marketed

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Chemical Groups of Germicides (cont.)

Cl: used for almost 200 years

Cl2 gas, OCl- (hypochlorite) and NH2Cl (chloramine) – all react with water to form hypochlorous acid (HOCl) – reacts with certain amino acids and denatures proteins

Disadvantages- relatively unstable… light, alkaline pH, and organic matter affect stability

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Chemical Groups of Germicides (cont.)I: Penetrates cells of microorganisms well

2-3% in water or 70% alcohol used a topical antiseptic for surgery5-10% for

Iodophores- I complexed to a polymer (e.g., polyvinylalcohol)… solves several problems…

-allows for slow release of I-increases penetration-less staining and irritating

e.g., Betadine

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Chemical Groups of Germicides (cont.)

2) Phenol derivatives (carbolic acid) – broad spectrum-derivatives referred to as phenolics- toxic to host cells – Lister (antiseptic surgery) – so not used much as antiseptics – disrupts cell walls, membranes and denatures enzymes

- all antimicrobials compared to phenol still (the phenol coefficient)-phenol used rarely but low percentages in soap solutions are commonly used (e.g., 1-3%, Lysol)- hexachlorophene - triclosan (Safeguard soap)

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Chemical Groups of Germicides (cont.)

3) Chlorhexidine – organic molecule with two phenol rings and Cl- targets cell membranes and dentures enzymes- bacteriocidal for Gram- and Gram+ organisms but is not sporicidal- low toxicity – used as obstetric antiseptic neonatal wash, wound antispetic, mucus membrane irrigant, preservative in eye solutions, and handscrubbing for doctors, preparing skin for surgical procedures

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Chemical Groups of Germicides (cont.)4) Alcohols - -OH functional groups

- ethyl and isopropyl are the only suitable candidates- Concentrations of >50% dissolve lipids- Dentures proteins as well in cytoplasm- Optimal concentration is 70% (water is required for

proteins to coagulate)- Removes oils on skin in which bacteria may be embedded

- Disadvantages – evaporates quickly, some organisms can use it, some toxicity with isopropyl

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Chemical Groups of Germicides (cont.)

5) H2O2 and related compounds- bacteriocidal (broad spectrum) and even sporicidal at high concentrations- action is through the hydroxyl free radical (OH-)- 3% H2O2 (aqueous) is routinely used for skin and wound cleansing and mouthwashing- can be a sterilant at 35%... Can get into parts of medical devices without corrosion - can be vaporized- Ozone (O3) can also be used as a disinfectant and works similarly to preoxide

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Chemical Groups of Germicides (cont.)6) Detergents and soaps

Detergents – have surface action (called surfactants) – are polar, charged molecules- most soaps fit here- cationic compounds work better than anionic ones… all have a long uncharged hydrocarbon chain (allows detergent to disrupt the cell membrane)- best example – quaternary ammonium compounds (quats) - benzalkonium chloride, cetylpyridinium chloride are commonly used- mixed with cleaning agents, used to disinfect floors, furniture, equipment surfaces… not good enough for medical devices (level of disinfection is low) - high concentrations – effective against some Gram+, viruses, fungi, and algae Low concentrations, mainly bacteriostatic

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Chemical Groups of Germicides (cont.)

Soaps: made up of fatty acids of oils with sodium or potassium (are salts of FA)

- Only weakly microbicidal and in fact, many bacteria including Pseudomonas aeruginosa can live and grow in soap dishes

- Removes oil on skin however and can be very effective at removing bacteria on skin if scrubbing accompanies the washing

- Thus many soaps add some other compound like I or chlorhexidine

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History of Chemotherapy• Folk Medicine (pre 1890’s)

– Plant products• Opium/morphine/heroin• Quinine Caffeine Cocaine• Salicin Digitoxin

• Diptheria antitoxin (1891)• Salvarsan for syphillus (1911)

– Arsenic compound– Paul Ehrlich– “magic bullets”

• Penicillium inhibited streptocci on petri plate (1928)– Alexander Flemming

• Protonsil Red (1935)– Streptococci– Gerhard Domagk– Sulphanomide

• Penicillin (1941)– Florey & Chain

• Streptomycin (1944)– Effective against

tuberculosis– Selman Waksman– Streptococcus griseus

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Chemical Groups of Germicides (cont.)7) Heavy Metals – Hg, Ag, Au, Cu, Zn, As have all been used at one time

or another for microbial control- Most too toxic to host!!! And can be absorbed through skin so even tough to use as disinfectants…may cause allergic reactions- broad spectrum as they bind and inactivate proteins - Mercurochrome- AgNO3 Solutions and newborns – are still some Ag-containing ointments particularly for burns

Some other disadvantages – Microbes can develop resistance to metals (genes for this often times on the same plasmids as those that have R factors!)- Large amounts of organic material (host wastes etc.) neutarlize action- Age generally weak as to their antiseptic qualities

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Chemical Groups of Germicides (cont.)8) The aldehydes – formaldehyde, and glutaraldehyde (have

–CHO functional group)- Intermediate to high level disinfectants

- “fixes” proteins- preserves tissues for many years…- Very toxic - Glut can be even used as a sterilant… killing spores in about 3 hours - “chemiclave” - vaporized glut- Formaldehyde gas – formalin (37% aqueous solution) – acts more slowly than glut - Formalin is a carcinogen

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Chemical Groups of Germicides (cont.)

9) Gaseous sterilants and disinfectants

a) Ethylene oxide-

b) Propylene oxide –

c) Chlorine dioxide -

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Chemical Groups of Germicides (cont.)

10) Dyes – even bacterial stains

- Crystal violet

-Malachite Green

11) Acids and Bases –

- Acetic acid –

12) Salt (NaCl) -

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Remember:Antiseptic and DisinfectingActivity is:

1) Concentration (of compound)2) Time3) Organism Dependent!!!

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Microorganisms & Antimicrobial Drugs• Antibiotics are common metabolic products of

aerobic bacteria & fungi

– Streptomyces (an Actinomycete) & Bacillus

– Penicillium & Cephalosporium (fungi)

• Inhibit other microbes in the same habitat, antibiotic producers have less competition for nutrients & space

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Mechanism of Antimicrobial Action• Antimicrobial drugs may simply inhibit bacterial growth BACTERIOSTATIC

• Drugs may also actively kill bacteria - BACTERIOCIDAL

• All exert action by inhibiting particular aspect of cellular physiology

• Five primary aspects of cellular physiology are targets• Cell Wall Synthesis• Protein Synthesis• DNA/RNA Synthesis• Maintenance of Plasma Membrane• Synthesis of Essential Metabolites

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Essential Metabolite

Selective Toxicity

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Inhibition of Cell Wall SynthesisPeptidoglycan

-lactam

Bulging surface of cocci

Weak points in peptidoglycan

25BSCI 424 PATHOGENIC MICROBIOLOGY U of Maryland

Page 352

-lactams

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Peptidoglycan• Macromolecule composed of a

repeating framework of long chains cross-linked by short peptide fragments– Unique to Bacteria– Composed of 2 sugars: NAG &

NAM– Sugars alternate in the backbone– Rows linked by polypeptides

• Provides strong, flexible support to keep bacteria from bursting or collapsing because of changes in osmotic pressure

N-acetylglucosamine(NAG)

N-acetylmuramic acid(NAM)

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Intact peptidoglycanNAM & NAG glycans cross linked by peptide bridges

-lactam antibiotics block peptidases that link the cross bridges between NAMs

PenicillinsCephalosporins

Relatively safe since mammals do not have peptidoglycan

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Some penicillins are less effective against Gram negative bacteria.Some cannot penetrate the outer membrane well.

Broad spectrum penicillins and cephalosporins can cross the cell walls of Gram negative bacteria.Carbenicillin & ceftriaxone

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Penicillins• All consist of 3 parts

– Thiazolidine ring– -lactam ring– Variable side chain dictates

microbial activity• R

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Neisseria gonorrhoeae

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Cephalosporins• Account for majority of all

antibiotics administered• Cephalosporium acremonium• -lactam• Relatively broad-spectrum• Resistant to most penicillinases

& cause fewer allergic reactions• Some are given orally, many

must be administered parenterally– Other than via the digestive tract

• Intravenous or intramuscular injection

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Vancomycin• Streptomyces orientalis

• Disrupt alanine-alanine bridges that link NAM in most Gram + bacteria– Bacteria lacking alanine-alanine bridges are

resistant

publications.nigms.nih.gov/.../chapter1.html

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Bacitracin• Peptide antibiotic

– In the product Neosporin

• Bacillus subtilis• Blocks movement of NAG and NAM from the

cytoplasm• Effective against Gram + • Topical antibiotic preparations

– Bacitracin has a high toxicity which precludes its systemic use

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• -lactams, vancomycin and bacitracin inhibit bacteria from building peptidoglycan

• Have no effect on already existing peptidoglycan

• Thus really most effective against actively growing cells

• Dormant cells or endospores are not susceptible

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Inhibitors of Specialized Cell Wall Synthesis• Unusual cell walls - Mycobacterium tuberculosis

• Isoniazid inhibits synthesis of mycolic acids… acid-fast stain…• Integral part of waxy cell wall• Essential for cell wall assembly

• Ethambutol inhibits incorporation of mycolic acid into waxy layer

• Both extremely effective against Mycobacterium

• Mycobacteria have generation times of 12 – 24 hours (slow growers)• Drugs administered over many months

37es.wikipedia.org/wiki/Imagen:Mycobacterial_cell_wall_diagram.png

Peptidoglycan

Plasma membrane

Cell wall skeleton

Outer lipids

Mycolic acid

Arabinogalactan L

ipoarabinomannan

Mycolic acids make up much of the cell wall of Mycobacterium

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Drugs that Block Protein Synthesis

• Ribosomes of eukaryotes differ in size and structure from prokaryotes

- 80s versus 70s – don’t forget subunit differences…– Antimicrobials selectively target bacterial

translation– Some of these drugs damage ribosomes in the

eukaryotic mitochondria or chloroplast• Contain a prokaryotic type of ribosome

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• Aminoglycosides – Insert on sites on the 30S subunit and interfere with the reading of the

codons on the mRNA• Tetracyclines

– Block attachment of tRNA on the A acceptor site and stop further synthesis

• Chloramphenicol– Attach to the 50S subunit – Prevents formation of peptide bonds

• Macrolides & Lincosamides – Attach to the 50S subunit– Inhibit translocation

• Movement of ribosome from one codon to another

• Oxazolidinones – Inhibits initiation complex formation– Binds to the 50S subunit and prevents the 30S complex from forming the

70S complex

Most of These Effect Some Part of Translation

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Antibiotics That Interfere With Protein Synthesis

Aminoglycosides – Gram- aerobic bacteria-Pseudomonas, Enterobacter, even Mycobacterium…

- not well absorbed through gut so usually administered intravenously and intramuscularly

Examples: Streptomycin, Neomycin, Gentamycin, Kanamycin

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Antibiotics That Interfere With Protein Synthesis

Tetracyclines: - Broad spectrum- First one was chlortetracycline (aureomycin) by Streptomyces aureofaciens… then Oxytetracycline by S. nimosus- Naturally-occurring versus synthetic

- Generally low toxicity… but Ca2+ problem… absorbed well though gut

- In 2005 tigecycline was developed… a new class of antibiotics called the glycylcyclines… first new tetracycline antibiotic in over 20 years… new ones are in clinical trials now

- Examples: Synthetic- Doxycycline, Minocycline

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Antibiotics That Interfere With Protein Synthesis

Macrolides - broad spectrum

- Used for upper resp. and soft tissue infections

- Used often as substitute for penicillin

- Toxicity low- absorbed well through gut

- Examples- erythromycin, azithromycin (Zithromax or Z-Pak)

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Antibiotics That Interfere With Protein Synthesis

Lincosamides – action like macrolides

- Lincomycin from S. lincolnensis- Used to treat Staph and Strept infections and also good

against some anaerobes, and protozoans such as Plasmodium (malaria)

- Other examples- Clindamycin (basically replaced Lincomycin)

- Clindamycin usually give intravenously

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Antibiotics That Interfere With Protein Synthesis

Oxazolidinones – Good against Gram+’s

- These were developed in the 90’s and used against MRSA when Vancomycin failed…

- First one developed was Linezolid (Zyvox)…

- Excellent oral bioavailability… and can be injected as well

- Other examples: AZD2563 (AstraZeneca)…shows great promise….

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Drugs that Disrupt Cell Membrane Function• A cell with a damaged membrane dies from disruption in

metabolism or lysis• These drugs have specificity for a particular microbial

group– Based on differences in types of lipids in their cell membranes.

• Polymyxins – Interact with phospholipids and cause leakage

– Originally the only class of antibiotic for Gram - bacteria

– Not effective against Gram + bacteria

– Topical use

• Toxic to kidney cells

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• Pyrazinamide– May inhibit fatty acid synthesis– Transport across the cytoplasmic membrane is

disrupted– Uniquely effective against Mycobacterium

• Mycobacterium accumulates this drug

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Inhibition of an Essential Metabolite• Sulfonamides and trimethoprim interfere with folate

metabolism– Block enzymes required for tetrahydrofolate synthesis needed for DNA

& RNA synthesis

• Competitive inhibition by sulfonamides– Structural analog– Drug competes with normal substrate for enzyme’s active site

• para-aminobenzoic acid (PABA)• Trimethoprim inhibits dihydrofolate reductase

– Next step in the tetrahydrofolate acid biosynthetic pathway

• Synergistic effect– Both drugs administered together– An additive effect, achieved by multiple drugs working together,

requiring a lower dose of each

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Sulphonamides inhibit dihydropteroate synthetase since it is a structural analogue of the normal substrate, PABA.

Trimethoprim inhibits dihydrofolate reductase, the next step in the folic acid biosynthetic pathway.

Humans convert dietary folic acid to THFA

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Quinolones Inhibit DNA Unwinding Enzymes

• Inhibit Gyrase– Introduces supercoils

• Page 258

– Essential for DNA replication– Necessary for packaging chromosome such that it

fits inside the cell

• Broad spectrum against Gram + & Gram -– Nalidixic acid – Norfloxacin – Ciprofloxacin

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Rifampin Inhibits RNA Polymerase

• Rifamycin inhibits prokaryotic RNA polymerase• Extremely soluble

• Can penetrate many tissues other drugs cannot

• Tuberculosis

• Meningitis

Antiviral Agents

• Inhibition of Viral Entry

• Inhibition of Nucleic Acid Synthesis

• Inhibition of Viral Assembly / Release

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Interferes with neuraminidase

Blocks entry

Prevents binding

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Inactivates DNA polymerase

Interferes with reverse transcriptase

Interferes with reverse transcriptase

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Interferes with viral protease

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Interferons• Signaling molecules made by virally infected cells

• Human proteins

• Secreted from infected cell, signals other cells to make anti-viral proteins (AVP)

• AVP produced in susceptible cells prevent viral replication

• Does not help cells that are already infected

• Only assists uninfected cells in resisting infection

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Mechanisms Drug Resistance• Drug inactivation

– Penicillinases– Genes on R plasmids– 200 different lactamases described

-lactamase

cephalsporinase

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• Decreased permeability to drug or increased elimination of drug from cell– Alteration of shape or charge of porins

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Multidrug resistant pumps• Self defense ejection system

• Lack selectivity & can pump out antimicrobials, detergents and toxins

publications.nigms.nih.gov/.../pump_it_up/

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• Change in drug receptors– Drug cannot bind to target

Vancomycin resistance develops when one D-alanine is converted to D-lactate

Instead of 3 H bonds now only two form between vancomycin and D-lactate

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• Change in drug receptor– Streptomycin resistance– A missense mutation changes the binding site

on the 30S subunit of bacterial ribosome

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• Change in metabolic patterns• Produce slightly different enzyme• Abandon metabolic pathway• Create a new metabolic pathway

Sulfonamide & trimethoprim resistance

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Preventing Drug Resistance• Complete the full antimicrobial prescription

– High concentrations of the drug must be maintained for a sufficient time to eliminate all sensitive cells

• Combinations– Synergistic– Some combinations are antagonistic

• Limit use– Estimated that 50% of the prescriptions for antibacterial drugs to treat

sore throats and 30% of prescriptions for ear infections are unnecessary

• Viral etiology• Drug Development

– Novel side chains to existing drugs– New drugs

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• Minimum Inhibitory Concentration (MIC)– Smallest concentration of drug that visibly inhibits growth

• Therapeutic Index – The ratio of the dose of the drug that is toxic to humans as

compared to its minimum effective dose

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Disk Diffusion Method• Spread dilute culture of bacteria over plate

• Place disk containing known amount antibiotic on plate

• Antibiotic diffuses out of disk, creates concentration gradient

• If bacteria is susceptible, a zone of inhibition is produced

• The larger the zone, the more sensitive the organism

• A qualitative test

• Sensitive

• Intermediate• Resistant

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Larger the zone, the greater the sensitivity to the antibiotic

Qualitative test

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Larger the zone, the greater the sensitivity to the antibiotic (must be comparable so the same antibiotic)

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Quantitative Sensitivity Tests• E-test is more quantitative

• Spread dilute culture over plate

• Place plastic strip with gradient of antibiotic

• Zone of inhibition is produced

• Can determine the minimum inhibitory concentration (MIC) of an antibiotic required for specific organism

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Broth Dilution Tests

• More versatile than E-test

• Allows determination of minimal bactericidal concentration (MBC) as well as MIC

• Make series of dilutions of antibiotic in growth media

• Inoculate each dilution with bacteria

• Determine minimum concentration required to inhibit growth

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But Remember- must reach that concentration in blood or in specific tissue!!