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Control of Microbial Populations I & II

MICR570/ZMR/F12 9-10

Control of

(Bacterial & Fungal)

Lectures 9 & 10

OBJECTIVES: LECTURE 9 & 10

• Overview Microbial Control

• Introduce key control methods – Preventing disease

– Treatment of disease

• Discuss key classes of therapeutic agents

• Introduce testing strategies

• Discuss Resistance – mechanism involved

Reduce numbers to

sanitary levels

AIMS OF CONTROL

inhibit

Eliminate/Kill

TERMINOLOGY

Biocides:

Disinfection: Disinfectant

– Destroy vegetative pathogens present on surfaces

– Not used on living tissues

Ant isepsi s: Ant isepti c

– destruction of vegetative pathogens on living tissue

Ant ibi ot ic = naturally-occurring (microbially-produced)

compound used in the treatment of disease – Also used to describe synthetic antimicrobials

Sterilization = removal of all life





# ’ s L o g 1 0

Add it ion of compound

“ -static” vs. “ -cidal”

static

M i c r o b i a l

Time

cidal

• Numbers & type of organisms: concentration of agent

(ppm, %, w/v)

FACTORS INFLUENCING CHOICE& OUTCOME

• Presence of organic material, E.g., blood pus

• Location of microbe/infection

• E.g., type of material/surface/object being treated

Exposure time

Amount/type of agent

bacteria

ResistantEndospores

Mycobacteria

Fungal spores

Prions

Small, non-enveloped viruses

Modified from Fig. 1.17, p.33. In: Antisepsis, Disinfection & Sterilisation. McDonnell, G. (2007) ASM Press

Sensitive

Gram negative bacteria

Vegetative fungi

Gram positive bacteria

Large, non-enveloped viruses

Enveloped viruses





• Numbers & type of organisms, concentration of agent

(ppm, %, w/v)

FACTORS INFLUENCING CHOICE& OUTCOME

• Presence of organic material, E.g., blood, pus

• Location of microbe/infection

• E.g., type of material/surface/object being treated

Exposure time

Amount/type of agent

bacteria

Keep in mind

• Aim of the treatment

• What is being treated

• Restrictions/limitations of the technique/chemical

agent

• Mechanisms of action

• Uses/applications

( A) Physical (B) Chemical

METHODS OF MICROBIAL

CONTROL

g empera ures:

Moist Heat

Pasteurization

Dry heat

Ethylene Oxide Gas

FiltrationLow temperatures

Radiation

oo preserva ves

Disinfectants

Antiseptics

Antibiotics

Antifungals

Antivirals

PHYSICAL CONTROLTEMPERATURE





Moist (Wet) Heat (boiling)

• Coagulates proteins: hydrogen bond broken

Native

protein

Coagulated

protein

heatComplex 3-D structure

EFFECTIVENESS OF HEAT

Temperature: 100oC

C botulinum endos ores

10 mins 5½ hours

.

Vegetative bacteria

Fungi

Time for inactivationImage: Zayaitz, A & Hussey, M. From: www.Microbelibrary.org

Dry Heat @ 121oC requires 600 mins

Autoclave = large pressure cooker

• Steam @ 1 atm pressure: 100oC

• Steam @ ≈2 atm pressure: 121oC

– 18-20 lbs/in2 (psi)

Steam under pressure:

• Will kill all organisms and most endospores in ≈ 15 mins

• Uses: culture media, solutions, dressings, instruments

NOT FOR HEAT-SENSITIVE ITEMS: SOLUTIONS,PLASTICS, etc

Sterilisation

© J. Rayner, 2008

Type Mechanism Applications

RadiationUV*

Convalent linkages

between DNA bases

Liquid, air & surface

disinfection

Ionizing*

(X-rays, γ-rays)

Causes release of

electrons

Disinfection & sterilization of

devices, cosmetics, water,

etc.

Ethylene

*

Alkylating agent Heat sensitive materials,

PHYSICAL CONTROL ALTERNATIVES

,

devices/equipment

Filtration Physical removal of

contaminants from

liquids & gases.

Heat-sensitive liquids

(vaccines, antibiotics)

Cold Freezing Prevention of growth

plus ice crystalformation (Ælysis)

Food preservation, long-

term culture storage, etc.

Refrigeration Reduce or prevent

growth

Preservation of lab media,

foods, etc.





ETHYLENE OXIDE gas

• Chemical sterilizing agent (used in gaseous form)

• Mechanism of action: strong alkylator Æ reacts with

guanine of DNA and functional groups of proteins

OHCH2

O

N

N

N

H2

OH

N

H

N

N

N

N

H2

OH

N

N

2

CH2

CH2

Guanine

CHEMICAL CONTROL

• Phenols, bisphenols &

Cresols

• Acids, acidulants & esters

• Biguanides

• Aldehydes

• Heavy metal derivatives

Types of chemical agents (disinfectants & antiseptics):

• a s

• Chlorine releasing agents

• Iodine & Iodophors

• Surface-acting agents

• Permeabilising agents

• Peroxygens

Wide variety of formulations: soaps, gels, creams, lotions…..

Agents Bacter ia Mycobact eria Bacteri al Spor es Fungi Viruses

Disinfectants

Alcohol + + - + +/-

Hydrogen peroxide + + +/- + +

Formaldehyde + + + + +

Phenolics + + - + +/-

Chlorine + + +/- + +

Iodophors + +/- - + +

Murray, Table 8-4, page 82, 6th Edition

Glutaraldehyde + + + + +

Quaternary ammoniumcompounds

+/- - - +/- +/-

Antis eptic Agents

Alcohol + + - + +

Iodophors + + - + +

Chlorhexidine + + - + +

Parachlorometaxylenol +/- +/- - + +/-

Triclosan + +/- - +/- +

Mechanisms of action

Reactions

affecting cell

components

Reactions:

Hydrolysis

Oxidation

Alkylation,

etc

Reactions

affecting

proteins

Reactions

affecting

membranes

Antimicrobial

activity





DISINFECTANTS

Inactivates: viruses,

fungi, mycobacteria &

spores

Risk &

disinfectant type

Part 2 Protocols - sterilization, disinfection and cleaning of medical equipment : guidance on decontamination from the Microbiology

Advisory Committee to Department of Health, Medical Devices Agency . 18th April 2005

,

fungi & mycobacteria

Inactivates: non

sporulating bacteria and

lipid-enveloped viruses

Disinfectant Action

Gluteraldehyde Cross-linking

Peracetic acid Oxidising agents

High

coagulation

Iodophors Oxidising agents

QACs (Quats) SurfactantsLow

ANTISEPTICS

• Commonly used as components in soaps,scrubs, sprays, gels…

• Effectiveness determined by: – Microorganisms

– Level of toxicity to tissues

Examples of biocides widely used as skin

antiseptics and washes

Biocide Active against

Alcohols (60-92%)Bacteria, fungi, viruses,

Mycobacteria

*Chlorhexidine (0.4 - 4%)

, , ,

some viruses

Iodine & Iodophors (0.5 -10%)Bacteria, fungi, viruses,

Mycobacteria

Triclosan (0.1-2%) Bacteria, fungi*, Mycobacteria*

* static effect

Modified from Table 4.3., p. 154, Chapter 4: Antisepsis & Antiseptics. In: Antisepsis, Disinfection &

Sterilisation. McDonnell, G. (2007) ASM Press





SPECTRUM OF ACTIVITY

= range of bacteria/fungi against which a compound is active

• Narrow

– Limited number of bacterial species (E.g., Metronidazole:

THERAPEUTIC ANTIMICROBIALS

• Broad

– Wide range of bacterial species (E.g., Aminoglycosides:

Gram positive, Gram negative, etc)

• Extended

– Usually refers to a Generation; increased number of susceptible

species compared to the previous generation

= compounds with minimal or no effect on host cells but

maximum effect against the infecting microorganism

• =

SELECTIVE TOXICITY

, . .,

– Peptidoglycan

– Ergosterol (antifungals)

• Alternatively: targets that are suitably different compared

to the host cell equivalent

Source: Natural or synthetic

ANTIBIOTICS

http://botit.botany.wisc.edu/toms_fungi/images/pen-colony.jpg

http://soils.usda.gov/sqi/concepts/soil_biology/images/SSSA14_LR.jpg

Considerations influencing choice

Immune status

of patient

Side effects

Cost

Degree of penetration Sensitivity of the org’

Site of Infection

Use of combination therapy

Local environment





Murray,

Figure 20-1p.202. 6th

Edition

3. Nucleic acid synthesis

1. Cell wall

2. Protein synthesis

4. Metabolism

1. Cell Wall Synthesis Inhibitorsβ-lactams

Glycopeptides

Bacitracin

Cycloserine

Picture taken from Brock Biology of Microorganisms, 11th Ed.

Peptidoglycan cell wall synthesis

& antibiotic inhibition

http://www.microbelibrary.org/images/spencer/spencer

_cellwall.html

Generations

Natural penicillinsE.g., Benzyl penicillin

Penicillinase-resistant penicillinsE.g., Methicillin

AminopenicillinsE.g., Ampicillin

Extended spectrum penicillinsE.g., Piperacillin

Images from: Wikipedia (public domain)





2. Protein Synthesis Inhibitors

Target site Mechanism

30S ribosomal subunit

Aminoglycosides Induce codon misreading

e racyc nes oc n ng o am noacy a e o

site

50S ribosomal subunit

Macrolides

Lincosamides

Streptogramins

Inhibit transpeptidation & translocation

Target binding at A & P sites

Inhibit peptide bond formation

LIMITATIONS ON USE

Group NOT

effective

against

Reason

Aminoglycosides Anaerobes Oxidative phosphorylation

absent in Anaerobes

ycopep es ram –ve arge s ze = no pene ra on

into cell

Nitroimidazoles Aerobes Requires activation by

flavodoxin (absent in

aerobes)

Penicillins

Cephalosporins

Mycoplasmas

Mycobacteria

Lack cell wall or

cell wall impenetrable

Number A + B

Addition of antibiotic Antagonism

ANTIBIOTIC COMBINATIONS

A + B

Time

of

bacteria

A

B

Synergy

Can also have indifference (i.e., no change)

Example 1.

PABA

Synergist ic Example:

Sulfamethoxazole & Trimethopr im(4 & 3. Metabolism & Nucleic acid synthesis inhibit ors)

Dihydropteroate synthetase

SulfamethoxazolePABA

Dihydrofolic acid

Tetrahydrofolic acid

Dihydrofolate reductaseTrimethoprim

Sulfonamide

*Enterococci: exogenous folic acid




MICR570/ZMR/F12 9-10/

LABORATORY SUSCEPTIBILITYTESTING

Basis for testing:

• Bacterial susceptibility varies

• Ensures correctly targeted treatment

•

• Minimizes cost

• Eliminates possibility of antagonistic effects (combination

therapies)

Testing provides an indication of the likely outcome of treatment

MIC = 1st tube WITHOUT

visible growth

1.6 μg/ml

QUANTITATIVE: Broth dilutionmethod: MIC & MBC

Plate out onto agar

MBC = plate

without visible

colonies

QUALITATIVE: Kirby-Bauer “ disk-

diffusion” method

http://en.wikipedia.org/wiki/Image:KB_test.jpg *See Lab Materials

E-test stripsalternative to MIC Macrodilution method

http://www.cdc.gov/ncidod/EID/vol12no08/images/06-0291_t.gif




MICR570/ZMR/F12 9-10/

The "down-side" of antibiotic treatment

‘…the greatest possibility of evil in self-medication is the

use of too small doses so that instead of clearing up

infection, the microbes are educated to resist penicillin

and a host of penicillin-fast organisms is bred out which

RESISTANCE

others until they reach someone who gets septicaemia or

a pneumonia which penicillin cannot save.’

The use of antibiotics does not “ create” resistance - it

selects for resistant microorganisms already present

withi n a population and enables them to dominate

S ir A l e x a n d e r F l em i n g , N e w Y o r k T i m e s , 2 6 J u n e 1 9 4 6

Figure 2: Effect of selective antibiotic pressure in bacteria. Mulvey, M & Simor, A.E.

(2009) CMAJ 180 (4)

YOU, as a healthcare practit ioner can limi t the spr ead of resistance & selection of resistant strains:

1. Wash your hands thoroughly between patient visits

2. Do not give in to patients' demands for unneeded antibiotics

3. When ossible rescribe antibiotics that tar et onl a. ,narrow range of bacteria

4. Isolate hospital patients with multidrug-resistant infections

5. Familiarise yourself with local data on antibiotic resistance

Alliance for the Prudent Use of Antibiotics

http://www.tufts.edu/med/apua/Practitioners/healthcare.html http://www.wiley.com/college/pratt/0471393878/student/activities/bacterial_drug_resistance/index.html




MICR570/ZMR/F12 9-10/

bacterium

Active β-lactam antibiotic

β-lactamases & β-lactamase inhibitors

β-lactamase producti on

Inactive

β-lactamase inhibitor

E.g., clavulanic acid

Act ive

β-lactam

antibiotic

bacterium

Figure 1: Sites of action and potential mechanisms of bacterial resistance to antimicrobial agents.

Mulvey, M.R. & Simor, A.E., CMAJ, Feb 17, 2009, 180 (4), p408-415.

Resistance β-lactams Glycopeptides Tetracyclines Aminoglycosides

Drug inactivation ++ + ++ Altered uptake + - +

COMMON/UNCOMMON

RESISTANCE MECHANISMS

Altered target + + -

- rare/occurs infrequently; + common; ++ very common (many sp.)

Location of resistance genes:

1. Chromosomal (E.g., mutation)

2. Transmissible plasmids

3. Transposable elements (E.g., transposons)

ACQUISITION & TRANSFER OF ANTIBIOTIC RESISTANCE GENES

Also important in transfer of range of other virulencegenes, E.g., for toxin production, etc

Mechanisms of transfer 1. Plasmids = CONJUGATION

2. Loose DNA = TRANSFORMATION

3. Bacteriophage = TRANSDUCTION

4. Jumping genes = TRANSPOSITION




MICR570/ZMR/F12 9-10/

Maintenance of new information

Important concept of genetic transfer

For bacteria to keep the genetic information gained

Consequences for new information

. egra a on

2. Stably maintained

3. Incorporation into chromosome

For maintenance require 2 or 3 (known as Recombination)

RECOMBINATION

= breaking and rejoining of DNA in new combinations

Types of recombination

1. Hom ologou s (general) – Conjugation, Transformation &

Transduction

2. Non-homologous (site-specific)

“Cut and paste” mechanism - Transposition

Linear DNA fragment Excised chromosomal fragment

OVERVIEW OF HOMOLOGOUS

RECOMBINATION

chromosome

Integrated

fragment

HOMOLOGOUS: between similar/identical DNA

F+ cell

(donor)

CONJUGATIONIntegrated plasmid

(episome) integrates

into recipient DNA

F- cellÆ F+ cell

PenicillinSÆPenicillinR

• tra genes

• PenicillinR

Image from Murray et al. Figure 3-14. Mechanisms of bacterial gene transfer. p35




MICR570/ZMR/F12 9-10/

Event Outcome

Complete transfer of F plasmid New F+ cell

Incomplete transfer Cell remains F-

omp e e rans er o p asm

and its integration into the

chromosome

r ce g requency

Recombination)

Integrated F plasmid initiates

transfer to new F- cell

Transfer of F plasmid plus

some or all of chromosome

COMPETENT CELL

E.g: Haemophilus, Streptococci

TRANSFORMATION

2nd strand: recombination & incorporation

DNA binding protein

1 strand degraded

Image adapted from Murray et al. Fig 3-14. Mechanisms of bacterial gene transfer. p35

TRANSDUCTION

Types of bacteriophage:

A. Vir ulen t (l yt ic): death of cell by lysis, releasing new phage

B. Temperate: Can switch between virulent/lytic phase &

Lysogeny = when bacteria are carrying a prophage

– Lysogeny

• Gene expression repressed (no phage genes

expressedÆ no new phage)

• Phage gene expression and replication triggered by

certain conditions

Bacterial chromosome breaks up

Phage DNA produced

Lytic Phage

attaches to

bacteria

Injects

DNA

Bacterial DNA

Generalized Transduction

Defective phage

Bacterial DNA

Introduced DNA incorporated into

chromosome(NO new phage produced)

Cell lyses; new phage

released

ro uc on o p age

heads & tails

Phage attaches &

injects DNA

phage




MICR570/ZMR/F12 9-10/

Phage integrates into

chromosome

TemperatePhage attaches

to bacteria

Injects

DNA

Bacterial DNA Maintains stablerelationship

Switches intolytic mode

Specialized Transductio n

Introduced DNA incorporated into

chromosome(NO new phage produced)

Defective phage

Phage attaches &

injects DNA

Phage DNA & bacterial DNA (from

near where phage was integrated)

new

hostcell

TRANSPOSITION

Transposons (Tn)/Insertion sequences = jumping genes

• Can move around within the cell:

– ChromosomeÆ Plasmid

– ChromosomeÆ Chromosome

How do transposons move around within these sites?

= non-homologous recombination: site-specific

recombinases (transposase)

• Transposon may then be transferred via mechanisms

discussed

Insertion Sequence (IS)

Simple transposon

IS

Inverted repeat | Transposase | Inverted repeat

No selectable genes

Genes for e.g., PenicillinR

Antibiotic resistance or other

trait

IS IS

Complex/Composite transposon

ANTIFUNGALSFig. 70-1 Sites of action of antifungal agents. p704

44

11

3322




MICR570/ZMR/F12 9-10/

4 broad categories of antifungals

1. Polyenes

2. Nucleic acid synthesis inhibitors

3. Ergosterol biosynthesis inhibitors

. c nocan ns

1. Direct Membrane Damage:Polyenes, E.g., Amphotericin B

ions

lipids

Bind to Ergosterol

ions

Cell

lyses

Broad spectrum Fungicide

5-FC

Fungal Cytosine deaminase

5-fluorouracil

2. Nucleic acid synthesis

inhibitor/Antimetabolite

c tosine

Incorporation into RNA

Inhibition of protein synthesis

Inhibition of thymidylate

synthetase

Inhibition of DNA

synthesis

5-fluorouridine monophosphate

5-fluorouridine tiphosphate

5-fluorouridine triphosphate

5-fluorodeoxyuridine

monophosphate

Redrawn from: J. Antimicro.Chemo. 2000, 46, p.171.

Figure 70-4

p. 7063. Ergosterol biosynthesis inhibitors

a. Azoles

E.g., Fluconazole

Fig. 70-4.




Inhibit squaline epoxidase

Accumulation of squaline

b. AllylaminesE.g. Terbinafine

No Lanosterol formed

Nor Ergosterol formed

Increased membranepermeabilityBroad spectrum

4. β-Glucan Synthesis Inhibito rs

Echinocandins

E.g., Caspofungin

= relatively new class of antifungals

• Block (1,3)-β-D-glucan synthetase

Nikkomycin Z – currently under investigation in

terms of therapeutic potential

• Prevents synthesis of chitin

• Competes with UDP-N-acetylglucosamine for

chitin synthase

ANTIFUNGAL RESISTANCE

Factors Contributing to

• Mutations in cytosine deaminase

• Decreased rate of transport into fungal cell

• era on o arge enzyme .g., mu a on, over-

expression)

• Alteration of Ergosterol biosynthetic pathway

• Growth as biofilm

In Summary -Control of Microbial

Populations

1. Variety of physical and chemical methods for microbial

control

.

microbial properties contribute to appropriate choice of

method/agent

3. Efficacy of biocides and anti-infectives varies widely

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