26.1 heat sterilization sterilization –the killing or removal of all viable organisms within a...

26.1 Heat Sterilization

• Sterilization– The killing or removal of all viable organisms

within a growth medium

• Inhibition– Effectively limiting microbial growth

• Decontamination– The treatment of an object to make it safe to

handle

• Disinfection– Directly targets the removal of all pathogens, not

necessarily all microorganisms

© 2012 Pearson Education, Inc.


• Heat sterilization is the most widely used method of controlling microbial growth

• High temperatures denature macromolecules– Amount of time required to reduce viability tenfold

is called the decimal reduction time

– Some bacteria produce resistant cells called endospores

– Can survive heat that would rapidly kill vegetative cells



• The autoclave is a sealed device that uses steam under pressure (Figure 26.3)– Allows temperature of water to get above 100C– Not the pressure that kills things, but the high

temperature

• Pasteurization is the process of using precisely controlled heat to reduce the microbial load in heat-sensitive liquids– Does not kill all organisms, so it is different than

sterilization


Figure 26.3Chamberpressuregauge

Steamexhaustvalve

Door

Thermometerand valve

Steam supplyvalve

Steam enters here

Steam exhaust

Jacket chamber

Air exits through vent

Total cycle time (min)

Tem

per

atu

re (

C)

Autoclave time

Stop steam

Beginpressure

Flowingsteam

Sterilization time

Temperatureof object beingsterilized

Temperatureof autoclave


26.2 Radiation Sterilization

• Microwaves, UV, X-rays, gamma rays, and electrons can reduce microbial growth

• UV has sufficient energy to cause modifications and breaks in DNA– UV is useful for decontamination of surfaces

– Cannot penetrate solid, opaque, or light-absorbing surfaces


Figure 26.4



• Ionizing radiation– Electromagnetic radiation that produce ions and

other reactive molecules

– Generates electrons, hydroxyl radicals, and hydride radicals

– Some microorganisms are more resistant to radiation than others



• Sources of radiation include cathode ray tubes, X-rays, and radioactive nuclides

• Radiation is used for sterilization in the medical field and food industry– Radiation is approved by the WHO and is used in

the USA for decontamination of foods particularly susceptible to microbial contamination

• Hamburger, chicken, spices may all be irradiated


26.3 Filter Sterilization

• Filtration avoids the use of heat on sensitive liquids and gases

– Pores of filter are too small for organisms to pass through

– Pores allow liquid or gas to pass through

• Depth filters– HEPA filters

– Membrane filters

– Function more like a sieve


Figure 26.6


26.3 Filter Sterilization

• Membrane filters (cont’d)– Filtration can be accomplished by syringe, pump,

or vacuum

– A type of membrane filter is the nucleation track (nucleopore) filter


Figure 26.7


Figure 26.8


26.4 Chemical Growth Control

• Antimicrobial agents can be classified as bacteriostatic, bacteriocidal, and bacteriolytic


Figure 26.9

Total cell count

Viable cell count

Time

Lo

g c

ell n

um

ber

Lo

g c

ell n

um

ber

Lo

g c

ell n

um

ber

Bacteriostatic Bacteriocidal

Bacteriolytic

Total cell count

Total cell count

Time

Time

Viable cell count

Viable cell count


26.4 Chemical Growth Control

• Minimum inhibitory concentration (MIC) is the smallest amount of an agent needed to inhibit growth of a microorganism – Varies with the organism used, inoculum size,

temp, pH, etc.

• Disc diffusion assay – Antimicrobial agent added to filter paper disc

– MIC is reached at some distance• Zone of inhibition

– Area of no growth around disc


Figure 26.10

Minimuminhibitoryconcentration


Figure 26.11

Nutrientagar plate

Discs containingantimicrobialagents are placedon surface

Inoculate platewith a liquidculture of a testorganism

Incubate for 24–48 h

Test organism showssusceptibility to someagents, indicated byinhibition of bacterialgrowth around discs(zones of inhibition)


26.5 Chemical Antimicrobial Agents for External Use

• These antimicrobial agents can be divided into two categories

– Products used to control microorganisms in commercial and industrial applications

• Examples: chemicals in foods, air-conditioning cooling towers, textile and paper products, fuel tanks

– Products designed to prevent growth of human pathogens in inanimate environments and on external body surfaces

• Sterilants, disinfectants, sanitizers, and antiseptics


III. Antimicrobial Agents Used In Vivo

• Antimicrobial drugs are classified on the basis of– Molecular structure

– Mechanism of action

– Spectrum of antimicrobial activity


Figure 26.12

CycloserineVancomycinBacitracinPenicillinsCephalosporinsMonobactamsCarbapenems

TrimethoprimSulfonamides

Quinolones

Cell wall synthesis

Folic acid metabolism

DNA gyrase

Nalidixic acidCiprofloxacinNovobiocin

Cytoplasmic membranestructure and function

PolymyxinsDaptomycin

THF

DHF

DNA

mRNA

Ribosomes

50 50 50

30 30 30

RNA elongation

Actinomycin

DNA-directed RNA polymerase

RifampinStreptovaricins

Protein synthesis(50S inhibitors)

Erythromycin (macrolides)ChloramphenicolClindamycinLincomycin

Protein synthesis(30S inhibitors)

TetracyclinesSpectinomycinStreptomycinGentamicinKanamycinAmikacinNitrofurans

Protein synthesis(tRNA)

Lipidbiosynthesis

MupirocinPuromycin

PlatensimycinCell wallCytoplasmic

membrane

PABA


Figure 26.13

Fungi

Eukaryotes Bacteria Obligately parasitic Bacteria Viruses

AzolesAllylamines

CycloheximidePolyenesPolyoxins

Nucleic acidanalogs

Echinocandins

Mycobacteria Gram-negativeBacteria

Gram-positiveBacteria

Tobramycin Penicillins

Streptomycin

SulfonamidesCephalosporins

Quinolones

Isoniazid Polymyxins

Tetracycline

VancomycinDaptomycin

Platensimycin

Chlamydia RickettsiaRNA

virusesDNA

viruses

Nonnucleosidereverse transcriptase

inhibitorsProtease inhibitorsFusion inhibitors

Nucleoside analogsInterferon


26.6 Synthetic Antimicrobial Drugs

• Paul Ehrlich studied selective toxicity in the early 1900s

– Selective toxicity is ability to inhibit or kill a pathogen without affecting the host



• Sulfa drugs: discovered by Gerhard Domagk in the 1930s– Inhibit growth of bacteria (sulfanilamide is the

simplest;

– Isoniazid is a growth analog effective only against Mycobacterium

• Interferes with synthesis of mycolic acid


Figure 26.16

Sulfanilamide

Folic acid

p-Aminobenzoic acid



• Nucleic acid base analogs have been formed by the addition of bromine or fluorine

• Quinolones are antibacterial compounds that interfere with DNA gyrase (e.g., ciprofloxacin)


Figure 26.17 Growth factor Analog

Phenylalanine(an amino acid) p-Fluorophenylalanine

5-Fluorouracil

5-Bromouracil

Uracil(an RNA base)

Thymine(a DNA base)


26.7 Naturally Occurring Antimicrobial Drugs: Antibiotics

• Antibiotics are naturally produced antimicrobial agents

– Less than 1% of known antibiotics are clinically useful

• Can be modified to enhance efficacy (semisynthetic)

• The susceptibility of microbes to different antibiotics varies greatly

– Gram-positive and gram-negative bacteria vary in their sensitivity to antibiotics

– Broad-spectrum antibiotics are effective against both groups of bacteria


26.8 -Lactam Antibiotics: Penicillins and Cephalosporins

• -Lactam antibiotics are one of the most important groups of antibiotics of all time– Include penicillins, cephalosporins, and

cephamycins– Over half of all antibiotics used worldwide

• Penicillins (Figure 26.19) – Discovered by Alexander Fleming– Primarily effective against gram-positive bacteria– Some synthetic forms are effective against some

gram-negative bacteria– Target cell wall synthesis


Figure 26.19

N-Acyl group

-Lactamring

Thiazolidinering

6-Aminopenicillanic acid

N-Acyl group Designation

NATURAL PENICILLIN

SEMISYNTHETIC PENICILLINS

Benzylpenicillin(penicillin G)

Methicillin

Oxacillin

Ampicillin

Carbenicillin

Gram-positive activity-lactamase-sensitive

acid-stable,-lactamase-resistant

acid-stable,-lactamase-resistant

broadened spectrum of activity(especially against gram-negativeBacteria), acid-stable,-lactamase-sensitive

broadened spectrum of activity(especially against Pseudomonasaeruginosa), acid-stable butineffective orally,-lactamase-sensitive


26.8 -Lactam Antibiotics: Penicillins and Cephalosporins

• Cephalosporins (Figure 26.20) – Produced by fungus Cephalosporium

– Same mode of action as the penicillins

– Commonly used to treat gonorrhea


Figure 26.20

Dihydrothiazinering

-Lactamring


26.9 Antibiotics from Prokaryotes

• Many antibiotics effective against Bacteria are also produced by Bacteria

– Aminoglycosides are antibiotics that contain amino sugars bonded by glycosidic linkage (Figure 26.21)

• Examples: kanamycin, neomycin, amikacin

– Not commonly used today• Neurotoxicity and nephrotoxicity• Considered reserve antibiotics for when other

antibiotics fail


Figure 26.21

Streptomycin Kanamycin

N-Acetyltransferase



• Macrolides contain lactone rings bonded to sugars (Figure 26.22)

– Example: erythromycin

– Broad-spectrum antibiotic that targets the 50S subunit of ribosome

• Tetracyclines contain four rings (Figure 26.23)– Widespread medical use in humans and animals

– Broad-spectrum inhibition of protein synthesis

– Inhibits functioning of 30S ribsomal subunit


Figure 26.22

Macrolidering

Sugars


Figure 26.23

Tetracycline analog R1 R2 R3 R4

Tetracycline

7-Chlortetracycline (aureomycin)

5-Oxytetracycline (terramycin)

H

H

HOH

OH

OHOH H

Cl

CH3

CH3

CH3



• Daptomycin (Figure 26.24)– Also produced by Streptomyces

– Used to treat gram-positive bacterial infections

– Forms pores in cytoplasmic membrane

• Platensimycin – New structural class of antibiotic (Figure 26.25)

– Broad-spectrum, effective against MRSA and vancomycin-resistant enterococci


Figure 26.24


Figure 26.25


26.10 Antiviral Drugs

• Most antiviral drugs also target host structures, resulting in toxicity

• Most successful and commonly used antivirals are the nucleoside analogs (e.g., AZT)

– Block reverse transcriptase and production of viral DNA

– Also called nucleoside reverse transcriptase inhibitors

• Nonnucleoside reverse transcriptase inhibitors (NNRTI) bind directly to RT and inhibit reverse transcription


26.10 Antiviral Drugs

• Protease inhibitors inhibit the processing of large viral proteins into individual components

• Fusion inhibitors prevent viruses from successfully fusing with the host cell

• Two categories of drugs successfully limit influenza infection:

– Adamantanes

– Neuraminidase inhibitors

• Interferons are small proteins that prevent viral multiplication by stimulating antiviral proteins in uninfected cells


26.11 Antifungal Drugs

• Fungi pose special problems for chemotherapy because they are eukaryotic (Figure 26.26)

– Much of the cellular machinery is the same as that of animals and humans

– As a result, many antifungals are topical

– A few drugs target unique metabolic processes unique to fungi


26.12 Antimicrobial Drug Resistance

• Antimicrobial drug resistance– The acquired ability of a microorganism to

resist the effects of a chemotherapeutic agent to which it is normally sensitive



• Most drug-resistant bacteria isolated from patients contain drug-resistance genes located on R plasmids

• Evidence indicates that R plasmids predate the antibiotic era

• The use of antibiotics in medicine, veterinary medicine, and agriculture selects for the spread of R plasmids (Figure 26.28)

– Many examples of overuse of antibiotics– Used far more often than necessary

(e.g., antibiotics used in agriculture as supplements to animal feed)



• Almost all pathogenic microbes have acquired resistance to some chemotherapeutic agents (Figure 26.29)

• A few pathogens have developed resistance to all known antimicrobial agents

– Methicillin-resistant S. aureus (MRSA)

• Resistance can be minimized by using antibiotics correctly and only when needed

• Resistance to a certain antibiotic can be lost if antibiotic is not used for several years


Figure 26.29

Gram-negativeGram-positive

Gram-positive/acid-fastFungus

Other gram-negative rods

Year

Candida albicans

Acinetobacter spp.

Enterococcus faecalis*

Streptococcus pneumoniae

Mycobacterium tuberculosis*

Haemophilus ducreyi

Salmonella typhi

Haemophilus influenzae

Neisseria gonorrhoeae

Pseudomonas aeruginosa*

Salmonella spp.

Shigella dysenteriae

Shigella spp.

Staphylococcus aureus


26.13 The Search for New Antimicrobial Drugs

• Long-term solution to antimicrobial resistance relies on the development of new antimicrobial compounds– Modification of current antimicrobial compounds

is often productive

– Automated chemistry methods (combinatorial chemistry) has sped up drug discovery

– 7,000,000 compounds must be screened to find a single useful clinical drug


26.13 The Search for New Antimicrobial Drugs

• Computers can now be used to design molecules to interact with specific microbial structures

– Most successful example is saquinavir• Binds to active site of HIV protease

• New methods of screening natural products are being used

– Led to the discovery of platensimycin

• Combinations of drugs can be used (e.g., ampicillin and sulbactam)

• Bacteriophage therapy© 2012 Pearson Education, Inc.

26.1 heat sterilization sterilization –the killing or removal of all viable organisms within a...

Documents

pearson education

use of heat

controlled heat

heatsensitive liquidsdoes

microbial load

microbial growthuv

pressure figure

gasespores of filter