Chapter 7 and 20

The Control of Microbial Growth

Antimicrobial Drugs

• Sterilization: Removal of all microbial life

• Sanitization: Lower microbial counts, reduce the number of

microorganisms to a safe level ( eating utensils)

• Disinfection: Removal of pathogens (applied to non-living objects)

• Degerming: Removal of microbes from a limited area of living tissue (the skin)

• Sepsis refers to microbial contamination.

• Asepsis is the absence of significant contamination.

• Antisepsis: Removal of pathogens from living tissue

• Aseptic surgery techniques prevent microbial contamination of wounds.

The Control of Microbial Growth -Terminology

• Biocide/Germicide: Kills microbes• Bacteriostasis: Inhibiting, not killing, microbes

• Alteration of membrane permeability• Damage to proteins• Damage to nucleic acids

Actions of Microbial Control Agents

Figure 7.1b

Bacterial populations die at a constant logarithmic rate.

Figure 7.1a

The Rate of Microbial Death

Table 7.2 Microbial Exponential Death Rate: An Example

Effectiveness of antimicrobial treatmentDepends on:

1. Number of microbes2. Environment

• The presence of organic matter

• Temperature - increased temperatures usually enhance the efficacy of antimicrobial treatment

–Time of exposure

–Microbial characteristics• resistance

• capsules

• biofilms

1. Heat– Thermal death point (TDP) – lowest temperature at which all the

microorganisms in a particular liquid suspension will be killed in 10 min– Thermal death time (TDT): Time it takes to kill a specific bacteria at a specific

temperature.

• Dry Heat Sterilization kills by oxidation– Hot air sterilization

– Direct flaming

• Moist Heat Sterilization - denatures proteins • Boiling or flowing steam

• Kills bacteria, fungi and viruses

• Less effective on endospores

• Autoclave - steam under pressure - denatures proteins • Kills endospores too

Physical Methods of Microbial Control

Hot-air Autoclave

Equivalent treatments 170˚C, 2 hrs. 121˚C, 15 min

sterilization indicators

Physical Methods of Microbial Control

• Pasteurization reduces spoilage organisms and pathogens. Equivalent treatments:

– 63°C for 30 min

– High-temperature short-time 72°C for 15 sec

– Ultra-high-temperature: 140°C for <1 sec

Physical Methods of Microbial Control2. Filtration

– Sterilization of liquids thru membrane with pore size 0.2 µm removes microbes

• Enzymes• Vaccines

– High-efficiency particulate air (HEPA) filters• removes microbes >0.3 µm• Lower the numbers of airborne microbes

3. Low temperature inhibits microbial growth– Refrigeration– Deep freezing– Lyophilization – freeze-drying

4. High pressure denatures proteins5. Desiccation prevents metabolism6. Osmotic pressure causes plasmolysis

7. Radiation damages DNA

– Ionizing radiation (X rays, gamma rays, electron beams)

• Use in pharmaceuticals, medical and dental supplies

– Nonionizing radiation (UV) • Control of closed environment

– Microwaves kill by heat; not especially antimicrobial

Physical Methods of Microbial Control

• Antiseptics - chemical agents used on living tissue

• Disinfectants - chemical agents used on inanimate objects

• Levels of chemical decontamination – High level germicides kill endospores, and if properly used, are

sterilants.

– Intermediate germicides kill fungal (not bacterial) spores, resistant pathogens such as tubercle bacillus, and viruses (used for non-invasive equipment).

– Low-level germicides kill vegetative bacteria and fungal cells, and some viruses (used for materials that may touch the skin, not mucous membrane).

Chemical Methods of Microbial Control

• Properties of the disinfectant – how it works determines what it will be effective against.

• Concentration of the disinfectant

• The degree of contact with microorganisms – if the surface needs cleaning or is porous microorganisms can escape contact with the disinfectant.

• Temperature should also be considered, since increased temperatures usually enhance the efficacy of disinfectants.

• Time left in contact is also an important consideration.

Principles of effective chemical decontamination

1. Halogens: non-metallic elements that commonly occur in minerals, seawater, and salts

– Chlorine, hypochlorite (Clorox), chloramines, iodine, iodophors

– Oxidizing agents (Bleach is hypochlorous acid (HOCl)) • Denaturation of proteins.

• Can be sporicidal.

2. Phenol (Phenolics) – Lysol, Triclosan: soap antibacterial additive– Damage cell membrane & precipitate proteins; bactericidal, fungicidal,

virucidal, not sporicidal.

– Active in organic matter.

3. Chlorhexidine - halogen and phenol compound– Disrupt plasma membranes

– Binds well to skin and mucus membranes

– Low toxicity

– Surgical hand scrubs and pre-op skin prep

– Not sporicidal

Chemical antimicrobial agents

Chemical antimicrobial agents

Table 7.6

4. Alcohols - Ethanol, Isopropanol– Denature proteins

– Dissolve lipids

– Denaturation requires H2O, which is

why aqueous preparations are better

than pure alcohols.

70% is best alcohol concentration

5. Hydrogen Peroxide- Weak (3%) to strong (35%)

- Produce highly reactive hydroxyls: (free radicals) that damage protein & DNA while also decomposing to O2 gas, toxic to anaerobes.

– Strong solutions are sporicidal

6. Detergents and soaps - Quaternary ammonium compounds (QUATS) - surfactants that alter membrane proteins of some bacteria and fungi.

– Not sporicidal

– Hospital cleaners

– Smells nice

– Soaps: mechanically remove dirt/soil and grease containing microbes

7. Heavy Metals - Ag (Silver), Hg (mercury), Cu (copper), and Zn (zinc) are used as germicidals.

– Oligodynamic action -When heavy metal ions combine with sulfhydryl (-SH) groups, proteins are denatured.

– Not sporicidal.

Chemical antimicrobial agents

8. Aldehyde - glutaraldehyde and formaldehyde – Kill everything by crosslinking/alkylating proteins & DNA.

– Glutaraldehyde in 2% solution (T.B.) used as sterilant for heat sensitive instruments.

– Formaldehyde: disinfectant, preservative, toxicity limits use.

9. Gases and Aerosols - Ethylene oxide, propylene oxide, betapropiolactone, and chlorine dioxide.

– Strong alkylating agents, sporicidal.

– Equipment, bedding, disposables, sterilization.

– Very toxic (eyes, skin, mucus, membranes). Carcinogenic? 10. Organic Acids - Used in foods to inhibit microbial growth.

– Sulfur dioxide, Sodium benzoate/benzoic acid, Sorbic acid, Propionic acid/Ca propionate, Nitrates and Nitrites

Chemical antimicrobial agents

Microbial Characteristics and Microbial Control

Figure 7.11

Evaluating a disinfectant

Evaluating a disinfectant

Figure 7.6

Disk-diffusion method

Learning objectives

• Define the following key terms related to microbial control: sterilization, disinfection, antisepsis, degerming, sanitization, biocide, germicide, bacteriostasis, and asepsis.

• Describe the patterns of microbial death caused by treatments with microbial control agents.• Describe the effects of microbial control agents on cellular structures.• Compare the effectiveness of moist heat (boiling, autoclaving, pasteurization) and dry heat.

• Describe how filtration, low temperature, high pressure, desiccation, and osmotic pressure

suppress microbial growth.• Explain how radiation kills cells.• List the factors related to effective disinfection.• Interpret results of disk-diffusion method.• Identify the methods of action and preferred uses of chemical disinfectants.• Identify the appropriate uses for surface-active agents.• Identify the method of sterilizing plastic lab ware.• Explain how microbial control is affected by the type of microbe.

Antimicrobial Drugs

• Chemotherapy - The use of drugs to treat a disease

• Antimicrobial drugs - A chemical substance that destroys pathogenic microorganisms with minimal damage to host tissues – Depending on the type of microbe the drug targets they can be

classified as antibacterial, antifungal, antiviral, or anti-parasitic.

• Antibiotic - Substance produced by a microbe that, in small amounts, inhibits another microbe

• Selective toxicity - A drug that kills harmful microbes without damaging the host

The History of Chemotherapy

• Paul Ehrlich developed the concept of chemotherapy to treat microbial diseases;

– He predicted the development of chemotherapeutic agents, which would kill pathogens without harming the host.

• 1928 – Fleming discovered penicillin,

produced by Penicillium chrisogenes.– 1940 – Howard Florey and Ernst Chain

performed first clinical trials of penicillin.

• Sulfa drugs came into prominence in the late 1930s.

Table 20.1

Antimicrobial drugs• Spectrum of antimicrobial drugs• The action of antimicrobial drugs

– General action is either by • directly killing microorganisms (bactericidal) • inhibiting their growth (bacteriostatic).

– Antibiotic activity• MIC - Minimal inhibitory concentration• MBC - Minimal bactericidal concentration

• Pharmacology• Blood concentration• Tissue distribution • Adverse reactions

– Antimicrobial agents should not cause excessive harm to normal microbiota.– Toxicity - Neuro, nephro and GI system– Hypersensitivity

• A superinfections results from a disruption of normal microbiota– when a pathogen develops resistance to the drug being used.– when normally resistant microbiota multiply excessively.

Figure 20.7

The Spectrum of Antimicrobial drugs• Antibacterial drugs affect different targets in a prokaryotic cell.

– Narrow-spectrum drugs affect only a select group of microbes - gram-positive cells, for example;

– Broad-spectrum drugs affect a more diverse range of microbes.

• Fungal, protozoan, and helminthic infections are more difficult to treat because these organisms have eukaryotic cells.

Figure 20.2 Major Action Modes of Antimicrobial Drugs.

2. Inhibition of protein synthesis: chloramphenicol, erythryomycin, tetracyclines, streptomycin

Transcription Translation

ReplicationEnzyme

ProteinDNA mRNA

3. Inhibition of nucleic acid replication

and transcription: quinolones, rifampin

1. Inhibition of cell wall synthesis: penicillins, cephalosporins, bacitracin, vancomycin

4. Injury to plasma membrane: polymyxin B

5. Inhibition of essential metabolite synthesis: sulfanimide, trimethoprim

Sites of action of antimicrobial drugs1. Inhibition of cell wall synthesis

– can inhibit NAG and NAM synthesis or crosslinking– peptidoglycan not found in humans– only affects actively growing cells: new walls– bacteriostatic– gram negatives less susceptible– Examples:

• Penicillin is the classic example of an inhibitor of cell wall synthesis• Ampicillin, bacitracin carbapenems cephalosporin • Methicillin, oxacillin, vancomycin

The bacterial cell lysing as penicillin weakens the cell wall.

Rod-shaped bacterium before penicillin.

2. Injury to plasma membrane– alter permeability – toxicity risk - all membranes similarity– Example: polymixin B

The action of antimicrobial drugs

The action of antimicrobial drugs3. Inhibition of proteins synthesis

– eukaryotes = 80s ribosome, prokaryotes = 70s

– can target 70s but toxicity to mitochondria

– inhibit peptide bond formation, attachment of tRNA, or cause misreading of mRNA

– broad spectrum

– Examples: streptomycin, tetracycline, chloramphenicol

The action of antimicrobial drugs

4. Inhibition of nucleic acid synthesis– Interfere with DNA replication or transcription (RNA synthesis)

– Examples:    the quinolines,   rifampin

5. Competitive inhibitors of the synthesis of essential metabolites– competitive inhibitor of enzyme in metabolite synthesis pathway

– broad spectrum

– Examples:  sulfonamides

Figure 20.13 - Overview

Antimicrobial drugs activity

Figure 20.17

• Disk-Diffusion Test (Kirby-Bauer test) is used in determining what treatment of antibiotics should be used when treating an infection– to determine the inhibition of bacterial growth measured under standard

conditions – method:

• a culture medium - the Mueller-Hinton agar• plate lawn of bacteria• place disks of known antimicrobials on lawn • measure zones of inhibition - The measurement

is compared to the criteria set by the National

Committee for Clinical Laboratory Studies (NCCLS).  • compare to a standard table for that drug

– Sensitive

– Intermediate

– Resistant

Antimicrobial drugs activity

Figure 20.18

• E Test– to estimate MIC (minimal inhibitory concentration) for effective dose

Antimicrobial drugs activity

Figure 20.19

• Broth Dilution Test– to estimate MIC (minimal inhibitory concentration)

– to estimate MBC (Minimal bactericidal concentration)

Antibiotic Resistance

• General mechanisms by which a microorganism may express resistance to an antibiotic

• All are consequences of microbial gene expression

• A variety of mutations can lead to antibiotic resistance.

1. Inactivation of the drug (Enzymatic destruction of the drug)

2. Prevention of penetration of the drug to the target site within the microbe (changes in plasma or other membrane permeability or formation of capsules)

3. Alteration of drug's target site (such as ribosomal proteins)

4. Rapid rejection of the drug

Antibiotic Resistance

• Resistance genes are often on plasmids or

transposons that can be transferred between bacteria.

• Improper use of antibiotics selects for resistance mutants.

• Improper use includes:– Using outdated, weakened antibiotics

– Using antibiotics for the common cold and other inappropriate conditions

– Use of antibiotics in animal feed

– Failure to complete the prescribed regimen

– Using someone else's leftover prescription

Figure 20.20

Antibiotic Resistance

Effects of Combinations of Drugs

• Synergism occurs when the effect of two drugs together is greater than the effect of either alone.

• Antagonism occurs when the effect of two drugs together is less than the effect of either alone.

The Future of Chemotherapeutic Agents• Antimicrobial peptides

– Broad spectrum antibiotics from plants and animals

• Squalamine (sharks)

• Protegrin (pigs)

• Magainin (frogs)

• Antisense agents– Complementary DNA short nucleic acids

that binds to a pathogen's virulence gene(s)

and prevents transcription

• siRNA– Complementary RNA that binds mRNA

to inhibit translation

Learning objectives• Name the microbes that produce most antibiotics.•  Describe the problems of chemotherapy for viral, fungal,

protozoan, and helminthic infections.• Define the following terms: spectrum of activity, broad-spectrum

drugs, superinfection.• Identify five modes of action of antimicrobial drugs.• List the advantages of each of the following over penicillin:

semisynthetic penicillins, cephalosporins, and vancomycin.• Describe how each of the following inhibits protein synthesis:

aminoglycosides, tetracyclines, chloramphenicol, macrolides. • Describe how rifamycins and quinolones kill bacteria.• Describe how sulfa drugs inhibit microbial growth.• Describe two tests for microbial susceptibility to

chemotherapeutic agents.• Describe the mechanisms of drug resistance