Microbiology 22 midterm 3

The ideal disinfectant 11

Kill all pathogens including endospores and viruses

Harmless to humans and animal tissues

Harmless to instruments and materials (noncorrosive, non-staining)

Good penetrating ability

Works well at cool temperatures

Nonflammable

Has cleaning ability

Has the deodorizing ability

Not inactivated by organic matter

Good shelf life

Low cost

Disinfectant reality 4

Very few achieve sterility

No single disinfectant is appropiate for all circumstances

Reducing microbial population or removing vegetative forms of pathogens

Disinfection is a gradual process; a disinfectant might need to be left on a surface for several hours

Dilution test 3

Current standard method by American Official Analytical Chemist

Common bacteria cultures used in this test, such as Staphylococcus aureus, E. coli, and pseudomonas aeruginosa

Procedures

• Dip a metal ring in a pathogen culture

• Dry ring at 37C for a short time

• Place the cylinder in a disinfectant/antiseptic at normal use dilution: 10 minutes at 20 C

• Place the cylinder in broth and incubate

• No growth = product worked

Disk diffusion method 2

Used to determine microbial susceptibility to disinfectant/antibiotics

Measure the zone of inhibition

Phenol 5

Carbolic acid

The first disinfectant/antiseptic, introduced by Joseph Lister

Denaturation of enzymes; disrupts cell membranes

Has few of the ideal characteristics

Rarely used now (irritates the skin and disagreeable odor)

Cresols 5

Phenol derivative, phenolics

The main ingredient in Lysol

Better in many ways than phenol

Disruption of plasma membrane

Very good surface disinfectants

Triclosan 7

Bisphenols

Active ingredient in many antibacterial soaps or detergents

Incorporated into kitchen cutting board and handles of knives and other plastic kitchenware

Inhibits an enzyme for the synthesis of lipid, mainly affecting the integrity of the plasma membrane

Effective against G+, yeast, some G-

Pseudomonas Aeruginosa, a Gram-, very resistant to triclosan

FDA bans its use in most products for home consumption (toothpaste exempted)

Alcohol 3 (5)

Effectively kill bacteria and fungi but not endospores and nonenveloped viruses

The mechanism of action is protein denaturation, dissolving lipids (lipid envelope of virus)

Can not be used to clean wound; cause coagulation of a layer of protein under which bacteria continue to grow

Ethanol

Isopropanol

Ethanol

Grain alcohol

Optimum concentration of ethanol is 70%

pure ethanol (no water) is not effective

Isopropanol

Rubbing alcohol

Works better than ethanol

Iodine 5

The oldest and most effective antiseptics against all kinds of bacteria, many endospores, various fungi, and some viruses

Impairs protein synthesis and alters cell membranes

Three forms: aqueous, tincture (solution in alcohol), and organic (iodophor)

Iodophor is effective as iodine but doesn’t stain; the most common commercial preparation is Betadine (povidone-iodine, commonly used for surgical preparation

Used for skin antiseptic and wound treatmet

Chlorine 4

Chlorine in water to form germicidal active group, hypochlorous acid (HOCl)

Oxidize the cellular enzymes

Clorox bleach, a disinfectant, contains sodium hypochlorite

In emergency for drinking water: add 2-4 drops of bleach to a liter of water; let it sit for 30 min

Heavy metals, Oligodynamic

Copper sulfate, Xgel, Silver sulfadiazine, Silver iodide, Zinc chloride

Copper sulfate

Combines with proteins; used as algicide in swimming pools

Xgel

copper containing hand sanitizer, alcohol-free

Silver sulfadiazine

topical cream for burns, wound dressings

SIlver iodide

surfacine, a surface antimicrobial

zinc chloride

mouthwash

Quaternary Ammonium Compounds (Quats)5

The most widely used surface-active agents; more effective against Gram+ + than Gram -

Fungicidal, amoebicidal, and virucidal against enveloped viruses; do not kill endospores or mycobacteria

The mode of action is unknown, may affect the plasma membrane and cause a change in cell permeability

Common quats: Zephiran and Cepacol

Can’t control the growth of Pseudomonas

Glutaraldehyde 6

Chemical relative of formaldehyde

Mechanism of action: inactive proteins

Disinfect endospores, respiratory therapy equipment in a hospital

Considered as a sterilizing agent

Bactericidal, tuberculocidal, virucidal, and sporicidal

Takes 3 to 10 hours to kill endospores

Chemical sterilization 3

Ethylene oxide (EO): is the gas most frequently used for sterilization at room temperatures.

It penetrates most materials and kills all microorganisms by protein denaturation

Larger hospitals often are able to sterilize even mattresses in special EO sterilizaers

Pathogens present from the udder

Tuberculosis

Listeria monocytogenes

Staphylococcus

Brucella spp. (brucellosis)

Pathogens present from fecal contamination

Salmonella spp.

Campylobacter sp.

E. coli

Pathogens present from personnel

Mainly a problem in hand milking

TB, diphtheria, streptococcus, etc.

Pasteurization

Minimum 72C for 15 seconds (75C if sugar in product), higher temperature often is used; this treatment known as HTST (high temperature short time)

Should remove all pathogens

Allowing only organisms that can withstand heat to survive:

• Streptococcus lactis

• Lactobacillus spp.

• Some Bacillus spp.

Ultra-high-temperature (UHT): 140 C for 4 seconds, sterilizing milk product/juice for long-term storage, e.g. coffee creamer

Disinfectants vs. antimicrobial drugs

Similarity: act by interfering with the growth of microorganisms

Difference: antimicrobial drugs must act within the host

The effect of drugs on cells and tissues of host are important

Chemotherapy

Use of chemicals (drugs) in treating disease (either infectious or noninfectious, e.g., cancer)

A term coined by Paul Ehrlich in searching for a ‘magic bullet’

‘Magic bullet’: a drug could hunt down and destroy a pathogen without harming the infected host

Ehrlich found a chemotherapeutic agent called salvarsan for treating syphilis in 1910, the first synthetic drug.

Selective toxicity

The basis of chemotherapeutic control of bacteria

The chemical being used should inhibit or kill the intended pathogen without seriously harming the host

Drug affects some aspect of the pathogen’s physiology that is not part of the host’s physiology; e.g. block an enzyme that only the pathogen has; block formation of cell wall (petitidoglycan)

Spectrum

Range of organisms affected by a drug

Broad spectrum: antimicrobial drug affects both gram-positive and gram negative, e.g. Tetracycline

Narrow spectrum: drug affects only one or the other, e.g. Penicillin G

Advantage and Disadvantage of broad spectrum

Advantage: more likely to affect an unidentified pathogen

Disadvantage: more damage to beneficial normal flora; greater chance of superinfection; infection by a second pathogen or the pathogen develops resistance to the drugs; i.e. overgrowth of Candida albicans

Inhibition of cell wall synthesis

Penicillin and others prevent the syntehsis of intact peptidoglycan -> cell wall weaken -> cell undergoes lysis

Selective Toxicity: human cells do not have peptidoglycan -> Penicillin has very little toxicity on host

Inhibition of protein synthesis

Antibiotics target bacterial 70S ribosomes either 50S subunit or 30S

Selective toxicity: eukaryotic cells have 80S ribosomes

• Adverse effects on host: Mitochondria in eukaryotic cells also have 70S ribosomes

Most drugs that inhibit protein synthesis have a broad spectrum activity, erythromycin is an exception

• Erythromycin does not penetrate the gram-negative cell wall, it affects mostly gram-positive bacteria

• Erythromycin is often used for people who have an allergy to penicillin

• Binds to 50S subunit

Injury to the plasma membrane

Change the permeability of the plasma membrane

Polymyxin B attaching to the phospholipids of the membrane causing disruption of the cell membrane

Some antifungal drugs combine with sterols in the fungal plasma membrane causing disruption of membrane

Selective Toxicity: animal cells have mostly cholesterol, not ergosterol, found in fungal cell membrane

Inhibition of nucleic acid synthesis

Drugs interfere with the processes of DNA replication and transcription

Some may interfere with host DNA as well

Exception: Rifampin with better selective toxicity

• It is a semisynthetic compound derived from Streptomyces

• It inhibits RNA polymerase in bacterial cells by binding its beta subunit (Quaternary), thus preventing transcription of messenger RNA and subsequent translation to proteins

• It provides much better selective toxicity

• It is typically used to treat tuberculosis and leprosy; and also used in therapy against Neisseria meningitidis (meningococcal) infection

Inhibition of synthesis essential metabolites

Drugs serve as competitive inhibitors for enzymes

Sulfonaminide (a sulfa drug) inhibits the synthesis of folic acid (functions as a coenzyme for synthesis of nucleic acids and amino acids) and stops the growth of the microorganism

Selective toxicity: humans do not synthesize folic acid, they obtain it in ingested food

Sources of antimicrobial drugs (3)

Synthetic drugs: produced in lab/factory by chemical reactions

• Ex. sulfa drugs

Botanical drugs: produced by plants

• Nearly 60% of the best-selling prescription drugs in America’s pharmacies are based on Mother Nature

Antibiotic drugs: substances of microbial origin having antimicrobial action

Antibacterial Drugs - sulfonamide (4)

A synthetic drug

Mode of Action: blocking the synthesis of folic acid

In bacteria, not in humans

Broad spectrum, a combination widely used

Antibacterial Drugs - Isoniazid (INH) (3)

A synthetic drug, very effective against Mycobacterium tuberculosis

Mode of Action: Inhibits the synthesis of mycolic acids; great selectivity

Combine use with other antimicrobial drugs, such as rifampin, to minimize the development of drug resistance

Antibiotics (5)

Substance produced by microbes; first discovered by Alexander Fleming in 1982 by accident

More than half of our antibiotics are produced by species of Streptomyces (fungus-like bacteria)

The function in nature: produced mainly by soil organisms to inhibit competing organisms

Commercial production: organism grown in large vat in appropriate broth; purified from the broth; biosynthesis (production of a chemical by a living organism)

Semisynthetic antibiotic: basic molecules produced by a microbe, then chemically modified

Vancomycin (5)

Glycopeptides antibiotics derived from a species of Streptomyces

Mode of action: inhibition of cell wall synthesis

Very narrow spectrum

Extremely important for treating MRSA (methicillin-resistant Staphylococcus aureus)

Lead to selection of Vancomycin-resistant enterococci (VRE): they are opportunistic gram-positive pathogens; considered a medical emergency

Tetracyclines (6)

Produced by Streptomyces

Broad-spectrum antibiotics; against both gram-positive and negative

Mode of action: inhibits protein synthesis

Penetrate body tissues well, very valuable against intracellular Rickettsias and Chlamydias

Suppress normal flora, lead to superinfection

Most common antibiotics added to animal feeds

Penicillin G (6)

Produced by mold Penicillum

Block cell wall formation

Works only during a cell division

Narrow but useful spectrum against gram-positive staphylococci, streptococci, and several spirochetes

Low toxicity, many allergies (humans), much resistance (bacteria)

Sensitive to penicillianases (beta-lactamases); are bacterial enzymes that destroy natural penicillins

Ampicillin; Amoxicillin (5)

Semisynthetic Penicillin

Part of the penicillin is produced by the mold, and part of added synthetically

Broad spectrum against many gram negative

Some resistance

Also senstiive to penicillinases (combined with inhibitor of penicillinase)

Oxacillin; methicillin (4)

Semisynthetic penicillin

Narrow spectrum against only gram-positive

Less resistance

Resistance to penicillinases

Quinine 6

antiprotozoal drugs

plant extract

treat malaria, but mostly resistant

first antimicrobial drug, bark of cinchona tree in 1630

blocks asexual reproductive cycle

synthetics chloroquine replaced

Antifungal drugs 5

difficult to find selective toxicity, physiology similar to host

target steros in plasma membrane, ergostral in fungi, chloesterol in animal

Azoles, most widely used antifungal drug group, e.g., imidazole

new class of drugs targeting cell wall, β-glucan

Echinocadins inhibit synthesis of glucans, imcpomlete cell wall and lysis, used to treat Candida spp.

Antiviral drug 5

Most antiviral drugs can’t dsetroy extracellular viruses or latent ones

60% illness by viruses, 15% bacteria, very few approved antiviral drugs

Possible modes of action, points in viral replication

• blocking synthesis of DNA or RNA; Serve as analogs of components of viral DNA or RNA; Purine and pyrimidine analogs, treating HIV and herpes

Acyclovir, azidothymidine (AZT)

Drug resistance 2

Pathogen is not affected by a drug

Pathogen changes, not drug or host

Selection 3

Everytime drug kills less than 100%

survivors are most drug resistant

Genetic variability in the initial population

Mutation (4)

Pathogen changes so its not affected by drug

Developes way to inactive drug, e.g. penicillianases

Prevent drug from reaching target site in pathogen

Target site changes, e.g., new enzyme appears that does same job but not affected by drug

Recombination

Drg resistance genes travel from pathogen to pathogen

Plasmid with resistance (R) factors contain R genes to several antibiotics

Rapid efflux (ejection)

pumps out drug before it becomes effective

gram - express numerous membrane transporters

agents can inhibit drug transporters can overcum efflux-associated resistance

Avoid unnecessary or inappropriate drug use (10)

Unnecessary: use for minor infections

Inappropriate: antibacterial for viral infection

sold w/o prescription

30% antibiotics prescribed for ear infection

100% for common cold

50% for sore throats unnecessary or not appropriate

use to full dosage

rotate drugs

minimise use of antibiotics in animal feed to promote growth

can take drugs off market for a year or more

Suusceptibility testing

determrin which crug might control an infection

disk diffusion method: kirby bauer test

Most widely used method

if effective, zone of inhibition forms around disk

reported as Sensitive, intermediate, or resistant

Effects of combination drugs

Synergism: 2+2=8

Antagonism 2+2=0

Drugs of future

Modify existing drugs to extend spectra of use and prevent resistance

Antimicrobial peptides

• produced by organiss other than microbes, like birds, frogs, ammals

• Magainins, produced by frogs, disrupt membranes, broad spectrum

Antisense agents

• Complementary DNA binds to pathogen virulence genes, prevents transcription

• Small interfering RNA (siRNAs): complementary RNA binds mRNA to inhibit translation