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CONTROL OF MICROBIAL POPULATIONS I & IIBACTERIA & FUNGI

Lectures 9 & 10

LEARNING OBJECTIVES At the end of these lectures, you should be able to:

1. Define the terms commonly used with respect to microbial control and be able toprovide examples of each.

2. Compare the physical control methods according to their mechanisms of action and

typical applications.

3. List the microbial and environmental factors that influence efficacy and use of a

particular control agent.

4. Differentiate between the non-therapeutic chemical control compounds (disinfectants

and antiseptics) based on cellular targets/mechanisms of action.

5. Contrast the challenges of treating fungal infections versus bacterial infections. 6. Distinguish between the major groups of therapeutic antimicrobials (antifungals and

antibiotics) on the basis of their cellular targets/activity by explaining the mechanisms

of action.

7. Characterise the mechanisms which bacteria evade the effect of antibiotics.

8. Describe the clinical microbiology laboratory tests for determining bacterial antibiotic

susceptibility/resistance.

9. Discuss the significance of inter and intra bacterial genetic transfer with regard to both

antibiotic susceptibility and virulence.

10. Contrast the mechanisms by which genes can be transferred between bacteria.

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INTRODUCTIONThe causative agents of infectious disease are controlled in four ways: by public sanitationmeasures, by sterilization and disinfection procedures, by chemotherapeutic agents and bythe body’s defensive mechanisms.

Chemotherapy did not begin until the early part of the 20th Century (See Introduction toMedical Microbiology Lecture) with the understanding of the principles of selective toxicity,specific chemical relationships between pathogens and drugs, development of drug resistanceand the role of combined therapy.

An understanding of the basic principles with respect to the use of an antimicrobial agent is anessential component of your knowledge, both for treatment and prevention of infection anddisease. Recognizing the rise in resistance to certain antimicrobial agents is essential tounderstanding the challenges you may face in the future.

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AIMS of CONTROL METHODSi. REDUCE microbial numbers to sanitary/acceptable levelsii. Slow down or INHIBIT microbial growth and replicationiii. KILL/ELIMINATE microorganisms

DEFINITIONSBiocide: a chemical agent that inactivates a wide range of microbes. Can be antiseptics,

disinfectants or preservatives.Disinfection: destruction/removal of vegetative pathogens from inanimate surfaces usingchemical or physical means. Antisepsis: killing or inhibition of microbial growth in or on living tissues usingnonchemotherapeuticagents. Antimicrob ials: naturally occurring or synthetic compound which inhibits or destroys selectivemicroorganisms, usually at low concentrations.Decontamination: reduction in microbial numbers so that the object is safe to handle.Germicide*: able to kill or inhibit growth and is sufficiently nontoxic to be used on livingtissues (*In some countries, refers to bactericidal activity only).Sterilization: a process that completely destroys or removes all microbial life including spores

NOTE: Compounds with the suffix:Static - inhibit the growth/replication of (bacteriostatic, fungistatic, sporostatic). Multiplicationresumes once agent removed.Cidal – have the ability to kill (bactericidal, fungicidal, sporicidal, virucidal). The action isirreversible.Lytic - dissolution of cells. Most lytic agents are cidal.

WHAT INFLUENCES CHOICE & EFFECTIVENESS OF AN AGENT?i. Type of microbe (See Diagram Below)ii. Numbers of microbe presentiii. Ability of the microbe to degrade or inactivate the agent

iv. Location (site) of the microbev. Physical environment (E.g., pH, temperature, humidity, etc.)vi. Concentration of the agentvii. Presence of organic material (E.g., blood, pus)

General Resistance to a BiocideMOST RESISTANT Prions

Bacterial SporesMycobacteria

Small, non-enveloped VirusesFungal Spores

Gram Negative Bacteria

Vegetative FungiLarge, non-enveloped Viruses

Gram Positive BacteriaLEAST RESISTANT Enveloped Viruses

Adapted from Microbiology: An Introduction, Tortora 8th Edition Pearson Education, Inc (2004)

METHODS OF CONTROL



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PHYSICAL CONTROL CHEMICAL CONTROLTemperature (high/low) Preservation (food) – Not discussedEthylene Oxide gas DisinfectantsFiltration AntisepticsRadiation Antimicrobials

PHYSICAL CONTROLNOTE: No given sterilization or disinfection procedure is applicable to every situation becausethe biological or physical nature of the object being treated differs from one situation to thenext.

TEMPERATURE

HEAT

WET DRYHeating: Pasteurisation & BoilingD IncinerationS

Steam @ atmospheric pressureD Hot air DS

Steam >atmospheric pressureS

S SterilisationD Disinfection

A common method that is relatively quick and inexpensive, having widespread use for controlling microbes on hospital instruments, lab media, food.Disinfection using heatDenatures and coagulates microbial proteins. Disassociates hydrogen bonds, renderingenzymes inactive → organisms KILLED.

Moist (Wet) HeatBoiling: 100oC (10 minutes will kill most but not all vegetative bacteria, fungi & viruses). Thisis ideal for safe food & water (NOT STERILE).

Pasteurization: For heat sensitive-liquids (milk) @71oC for 15 seconds (NOT STERILE).

Sterilisation using heatSteam under pressure (2atm) achieved by an autoclave (large pressure cooker) to increasetemperatures. @ 15 lbs/in2 (psi), temperature reaches 121oC. This will kill ALLmicroorganisms and most endospores in ≈15 minutes.Commonly used for culture media, dressings, instruments.

Moist heat has a better penetrating power than dry heat, producing a faster death rate at agiven temperature.

ALTERNATIVE METHODS OF PHYSICAL CONTROL (See Table 8-1, p80, Murray et al.)Type Mechanism ApplicationRadiationUV Cause convalent linkages

between DNA basesLiquid, air & surface disinfection



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Ionising Rays(X-rays, γ-rays)

Cause release of electrons Disinfection & sterilization of devices,cosmetics, water, etc.

Ethylene Oxide gas* Alkylating agent Heat-sensitive materials,plastics,complex devices/equipment

Filtration Physical removal of

microorganisms

Heat-sensitive liquids (vaccines,

antibiotics), gases, etc.ColdFreezing

Refrigeration

Prevention of growth plus icecrystal formation (lysis)

Reduce or prevent growth

Food preservation, long-termculture storage, etc.

Preservation of lab media,foods, etc.

*Can be used for sterilisation

CHEMICAL CONTROLGeneral term to describe chemical agents routinely used to control microbial growth, including

antiseptics, disinfectants, therapeutic antimicrobials, etc.

Types of chemical agents (See Table 8-4, p82, Murray et al.)

Mechanisms of Ac tioni. Affect Proteins• Denaturation – alteration of protein structure• H2 & S-S bonds changed – function of protein impaired• Permanent or Temporaryo Reactions affecting proteins

Hydrolysis Oxidation (E.g., peracetic acid, Iodophors)

Attachment of atoms or chemical groups (E.g., alkylating agents)

ii. Affect Membranes (Proteins & Lipids)• Proteins - affected by above reactions• Disruption of Lipids (E.g., surfactants (QAC’s), alcohols)

iii. Affect Other Cell Components• Nucleic acids & Energy production systems – alkylation (E.g., peracetic acid, gluteraldehyde,ethylene oxide)

DISINFECTANTS

Categories of disinfection (Summarised from Murray et al., p79-80)1. HIGH LEVEL: inactivate viruses, fungi, mycobacteria & bacterial spores.For items involved with invasive surgery (E.g., endoscopes).Examples: glutaraldehyde, H2O2, peracetic acid2. INTERMEDIATE LEVEL: inactivate viruses, fungi & mycobacteria.For surfaces where endospores/resistant bacteria is unlikely (E.g., vaginal specula).Examples: alcohols, iodophors

3. LOW LEVEL: inactivate non-sporulating bacteria and lipid-enveloped viruses.



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Considerations influencing choice

Immune status

of patient

Degree of penetration Sensitivity of the org’

Site of Infection Local environment

Side effects

Cost

For devices that do not penetrate mucosal surfaces or sterile tissues (E.g., stethoscopes).Examples: QAC’s – Quaternary Ammonium Compounds

ANTISEPTICSMost are used for handwashing or treating surface wounds. In certain circumstances someantiseptics are also effective disinfectants.

Desirable features of antiseptics:• Wide spectrum of activity• Rapid activity• Low level of damage, irritation or toxicity to tissue being treated• Some form of persistence (continued activity) after initial use

Examples of Applicationsi. Skin hygiene (reduces #’s of normal flora/contaminating microbes)ii. Reduce #’s of microbes on a mucous membrane or the skin before breach (i.e., surgery,injection, etc)iii. Treatment of skin or wounds infections

THERAPEUTIC ANTIMICROBIALSDiscussion of antibiotics and antifungals. PLEASE NOTE: antivirals will be covered in Controlof Viral Populations Lecture.

SPECTRUM OF ACTIVITYBroad-Spectrum: one that is active against a wide variety of bacteria (i.e., Gram +ve as wellas Gram –ve).Narrow-Spectrum: effective against specific families of bacteria (i.e., one group of bacteria or

one species).

SELECTIVE TOXICITY CONCEPTIDEALLY antimicrobials should have minimal or no effect on host cells but maximum effectagainst infecting microbe. This may be a function of specific receptor for agent attachment or depend on inhibition of biochemical activities of the pathogen.Think about UNIQUE TARGET SITES (Bacterial & Fungal)

Alternative: (if no targets) sites/receptors suitably different from the host equivalentBACTERIAL • •

• •

• •

FUNGAL • •

• •

•



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ANTIBIOTICSSource of Antibiotics: Natural or Synthetici. Actinomycetesii. Filamentous fungiiii. Bacteria, especially Streptomyces spp.

Antibiotics are usually metabolic end products (secondary metabolites).Penicillin originally from Penicillium notatum now produced synthetically.Some are completely synthetic E.g., Quinolones

Analogues of nalidixic acid with quinolone or derived ringFluoroquinolones: modification of original structure with different side chainsubstitutions

General Characteris ticsTHINK ABOUT THE IDEAL PROPERTIES FOR A CHEMOTHERAPEUTIC AGENT•

•

•

•2 important criteria are (1) agent must be antimicrobial at low concentrations & (2) should berelatively nontoxic.Considerations that can influence choice of an antibiotic for treatment include:

Basic Principle of Antibio tic TherapyUnderstanding physiological processes E.g., growth requirements (anaerobic) and basiccharacteristics E.g., Gram staining, can influence the choice of a compound for treatment.Reversibility of antibiotic binding may influence cidal vs. static effect. Some antibiotics havedifferent activities when used individually compared to in combination.

TARGET SITE/MECHANISM OF ACTION (See Fig 20-1, p202 & Table 20-1, p201 Murray et

al.)There are 5 basic groupings1. Inhibitors of cell wall synthesis2. Inhibitors of protein synthesis (i.e., inhibition of translation & transcription)3. Inhibitors of nucleic acid synthesis4. Inhibitors of cell membrane function5. Inhibitors of metabolismWe are not going to discuss these in detail, rather relate a couple of the groupings to bacterialcell structure (See Introduction to Bacteria Lectures).1. INHIBITORS OF CELL WALL SYNTHESIS



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A + B

Time

Number

of

bacteria

A + B

A

B

Addition of antibiotic

Synergy

Antagonism

ANTIBIOTIC COMBINATIONS

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

Example 1.

EXAMPLES: β-Lactams (E.g. Penicillins, Cephalosporins, etc.), Glycopeptides (E.g.,Vancomycin), Polypeptides (E.g., Bacitracin).

Steps in Peptidoglycan SynthesisPeptidoglycan monomers are synthesized in the bacterial cytosol, where they attach to amembrane carrier molecule (bactoprenol). Bactoprenols transport the monomers across thecytoplasmic membrane. Autolysins break the glycosidic bonds between the peptidoglycan

monomers at the site of growth along the existing peptidoglycan chain as well as the peptidecross-links. Transglycosidase enzymes then insert and link new peptidoglycan monomers intothe breaks in the peptidoglycan and transpeptidase enzymes reform the peptide cross-linksbetween the rows and layers of peptidoglycan.

β-LactamsLarge group of cell wall inhibiting compds.Shared features of all members:• Active only on GROWING bacteria (i.e., actively replicating)

o Interfere with precursor incorporation & cross-linking• β-lactam ring as part of basic structure

o Penicillins: β-lactam ring + 5 membered thiazolidine ring

o Cephalosporins: β-lactam ring + 6 membered dihydrothiazine ring• Inhibit transpeptidation (enzymes PBP’s) involved in cross-linking of peptidoglycansubunits into growing cell wall.

2. INHIBITORS OF PROTEIN SYNTHESISEXAMPLES: Aminoglycosides, Macrolides, Azalides, Ketolides, Lincomycins, Tetracyclines,Glycylcyclines, Chloramphenicol, Streptogramins, Oxazolidinones.N.B. Bacterial Ribosomes structure (See Introduction to Bacteria Lectures).There are a variety of target sites and mechanisms of action. Not all inhibitors use the sametarget or mechanism.

Recognise the Limitations on the use of certain antibiotics Antibiot ic group NOT effect ive against Reason for lack of effect Aminoglycosides: Anaerobes Oxidative phosphorylation necessary for uptake (absent in anaerobes)

Glycopeptides: Gram –ve Large size, prevents penetrationNitroimidazoles: Aerobes antibiotic has to be activated by flavodoxin (absent in aerobes)Penicillins, Cephalosporins : Mycoplasmas (lack cell wall); Mycobacteria (cell wall impenetrable)

ANTIBIOTICS USED IN COMBINATIONReasons for use:1. Delay emergence of resistant strains2. Increase the spectrum of activity

3. Increase effect of combination (synergy)

Disadvantages of combination use:1. Possibility of drug reactions2. Cost3. Rarely achieve synergy4. Antagonism between the two drugs

EXAMPLES



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Synergistic EffectNormal folic acid synthesis pathway - indicate targets for SMX and TMPSulfonamides: inhibit PABA for synthesis of folic acidTrimethoprim or Pyrimethamine: inhibit reduction of Dihydrofolic acid → Tetrahydrofolic acidPABA Dihydrofolic acid Tetrahydrofolic acidEffective for some infections E.g., Shigellosis and Salmonellosis

Antagonis tic EffectChloramphenicol: inhibits protein synthesis (bacteriostatic)Penicillin: inhibits cell wall synthesis (bactericidal)

Antagonism occurs when bacteriostatic drug reaches target site before cidal drug, particularlyif destroying bacteria is key to curing infection.

ANTIBIOTIC SUSCEPTIBILITY TESTINGBasis for testing:1. Ensures correctly targeted treatment2. Minimizes selection for resistance

3. Minimizes cost4. Eliminates possibility of antagonistic effects

QUANTITATIVE METHODBROTH DILUTION METHODTwo-fold (log2) dilutions of the antibiotic are made in liquid growth medium (Muller-HintonBroth) and inoculated with test bacteria. Following incubation, the tubes are examined visuallyor read with a machine. Identifies the Minimal Inhibitory Concentration (MIC) (i.e., the lowestconcentration of antibiotic at which there is no visible growth).Once the MIC is established can also determine the Minimal Bactericidal Concentration(MBC) (i.e., the lowest concentration of antibiotic at which no growth occurs on an agar plate).Samples from the MIC tubes with no visible growth are plated onto agar plates and incubated.

Reasons to perform an MICa. Patient is critically ill with a potential fatal infection (E.g., meningitis or endocarditis)b. Monitoring of resistance development during therapyc. Low level antibiotic resistance possible in the clinical isolated. Determine the activity of a new antimicrobial in vitro

QUALITATIVE METHODSKIRBY-BAUER (“ Disk-Diffus ion” ) METHOD

A fully standardised assay, most widely used test in clinical labs.Bacteria seeded onto Muller-Hinton agar plates, antibiotic-impregnated discs (measuredquantity) are placed onto the plate. Following incubation, the diameter of the clear zone of

inhibition of bacterial growth surrounding the disc is measured as the inhibitory power of theantibiotic against the test organism.Principle: As the distance away from the disc increases, the concentration of the antibiotic inthe agar decreases.Inhibition zones of clinical isolates are compared to standard tables showing zones of inhibition for a known species (See Laboratory Manual, p11-12).

E-TEST STRIPS Alternative to tube/microteter plate MIC’sPlastic strips containing the antibiotic concentration gradient on one side and a numerical



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scale indicating antibiotic concentration on the other are placed onto Muller-Hinton agar platesinoculated as for the disc diffusion method. Following incubation, the plate is examined andthe number point at which the border of growth inhibition intersects the E test strip is the MIC.

Used to provide MIC data in situations where the level of resistance can be clinically

important.

In vitro activity of some selected bacteria (modified from p.498, Bacteriology Volume 1, Topley& Wilson’s Microbiology & Microbial Infections).

Minimum Inhibitory Concentration (mg/l)

Bacterium Benzylpenicillin Erythromycin Ciprofloxacin Imipenem

S. aureus 0.1 0.25 0.5 0.03

E. coli 64 R 0.06 0.25

P. aeuginosa R R 0.25 1

B. fragilis 16 1 8 0.1

C. trachmoatis R 0.25 0.5 R

Use this data to:1. Identify general patterns of bacterial sensitivity or resistance to the various antibiotics givenin the table.2. Identify if there is a difference in antibiotic sensitivity according to cell wall composition (i.e.,Gram+ve vs. Gram –ve).3. Identify which of the pathogens listed is generally more sensitive and less sensitive to theantibiotics given in the table.

ANTIBIOTIC RESISTANCEThe down side of antibiotic treatmentEXAMPLES OF VALUABLE ANTIBIOTIC THERAPIES NOW LOST OR IMPERILLED BYTHE SPREAD OF RESISTANCE

Organism Disease Agents lost or threatenedStreptococcus pneumoniae Pneumonia, otitis, meningitis Penicillin; many othersNeisseria meningitides Meningitis, septicaemia Sulphonamides; (penicillin)Shigella spp. Bacillary dysentery Most relevant agentsNeisseria gonorrhoeae Gonorrhoea Sulphonamides, penicillin,

tetracyclineFull report available:http://www.parliament.the-stationery-office.co.uk/pa/ld199798/ldselect/ldsctech/081vii/st0701.html

WORRYING DEVELOPMENTSMRSA: Methicillin (Multiple) Resistant Staphylococcus aureusVRE: Vancomycin Resistant EnterococciVISA: Vancomycin Intermediate Staphylococcus aureusVRSA: Vancomycin Resistant Staphylococcus aureusESBLS: Extended Spectrum Beta-Lactamases

MECHANISMS OF RESISTANCE AVAILABLE:1) Enzymatic Inactivation: E.g., Staphylococci produce β-lactamases - resistance toPenicillin G2) Decreased Permeability: E.g., Streptococci possess natural permeability barrier to

Aminoglycosides.



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Direct uptake of donor DNA by recipient cell (natural or forced). DNA fragments (usually about20 genes long) from a dead degraded bacterium bind to DNA binding proteins on the surfaceof a competent recipient bacterium. Nuclease enzymes then cut the bound DNA intofragments. One strand is destroyed and the other penetrates the recipient bacterium. ThisDNA fragment from the donor is then exchanged for a piece of the recipient's DNA by meansof Rec A proteins.

Naturally competent transformable bacteria include: B. subtilis, H. influenzae, N. gonnorhoeae& S. pneumoniae.

3. TRANSDUCTION: via BacteriophageInvolves the transfer of fragments of DNA from one bacterium to another bacterium by abacteriophage.There are 2 phage types:i. Virulent (lytic): after phage replication, the infected bacterium dies (lysis) and the pahge isreleased.ii. Temperate: Ability to switch between virulent/lytic phase and where DNA phage is stablyintegrated within host bacterial DNA (“prophage”). Bacterium has undergone lysogeny and

phage gene expression is normally repressed.There are two types of transduction: generalized transduction and specialized transductionthat can be distinguished according to:• Location of bacteriophage in the cell• Material being transferred (i.e., bacterial DNA or hybrid bacterial/phage DNA)N.B. Phage head is of limited size, it cannot carry its own DNA and a bacterial chromosome.

Generalized transduction1. A lytic bacteriophage adsorbs to a susceptible bacterium.2. The bacteriophage genome enters the bacterium. The genome directs the bacterium'smetabolic machinery to manufacture bacteriophage components and enzymes.3. Occasionally during maturation, a bacteriophage head or capsid assembles around a

fragment of donor bacterium's nucleoid or around a plasmid instead of a phage genome bymistake.4. The bacteriophages are released.5. The bacteriophage carrying the donor bacterium's DNA adsorbs to a recipient bacterium.6. The bacteriophage inserts the donor bacterium's DNA it is carrying into the recipientbacterium.7. The donor bacterium's DNA is exchanged for some of the recipient's DNA.

Specialized transduction1. A temperate bacteriophage adsorbs to a susceptible bacterium and injects its genome.2. The bacteriophage inserts its genome into the bacterium's nucleoid to become a prophage.

3. Occasionally during spontaneous induction, a small piece of the donor bacterium's DNA ispicked up as part of the phage's genome in place of some of the phage DNA which remains inthe bacterium's nucleoid.4. As the bacteriophage replicates, the segment of bacterial DNA replicates as part of thephage's genome. Every phage now carries that segment of bacterial DNA.5. The new bacteriophage adsorbs to a recipient bacterium and injects its genome.6. The bacteriophage genome carrying the donor bacterial DNA inserts into the recipientbacterium's nucleoid.



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TRANSPOSITIONThe ability of genes to change position on chromosomes, a process in which a transposableelement is removed from one site and inserted into a second site in the DNA. “JumpingGenes”.

Transposon (Tn):Simple transposons: similar to Insertion Sequence (IS) elements. Contain DNA segments

flanked by short inverted repeat sequences. The DNA segments, usually code for a number of gene products. In addition to a transposase, they may also code for resolvase (depending onmechanism of transposition) and may contain for one or more antibiotic resistance genes.

Composite transposons: are DNA segments flanked by an IS element at either end. Instead of each IS element moving independently, they now act and move together along with theintervening DNA. Each IS element is typical, although only one of the two elements typicallyretains a functional transposase activity. The IS elements may be in the same or in theopposite orientation with respect to one another. The intervening segment often carries thegenetic determinants for a number of antibiotic or other toxin resistances

Mechanisms for transposon movement

Simple transposition (direct or conservative transposition) involves transfer from one place toanother. The original element is excised from the original site leaving a double-strand breakbehind. The original molecule is irreparably damaged as a result. At the same time, astaggered cut is introduced into a target site into which the element is ligated. This mechanismresults in the duplication of the target site sequence on each side of the inserted element.

Replicative transposition involves the element replicating itself from place to place. Thismechanism requires staggered cuts on either side of the mobile element and in the target site.The two molecules are now joined and repaired creating a cointegrate - essentially the twomolecules are fused together. However, we now have two copies of the mobile element in thesame molecule. They are oriented in the same direction, and so a site-specific recombinationreaction (catalyzed by resolvase) can now occur generating the two original molecules, each

with a copy of the mobile element.

ANTIFUNGALSIt can be difficult to treat fungal infections without toxic side effects to the patient WHY?LIMITATIONS OF USE• Profound side effects• Narrow antifungal spectrum• Poor penetration of certain tissues• Selection of resistant fungi

TARGET SITES (See Fig 70-1, p704, Murray et al. Sites of action of antifungal agents).

Most agents target either:i. Structure (E.g., cell wall or membrane)ii. Physiological function (E.g., enzyme/metabolic process)

CATEGORIES OF ANTIFUNGAL AGENTS (See Table 70-1, p702-3, Murray et al.)1) POLYENES (E.g., Amphotericin B)Broad spectrum, fungicidal. Derived from Streptomyces spp. Used for severe systemicmycoses.Mode of Action: Binds directly to ergosterol in cell membrane, resulting in membrane damage



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(altering membrane fluidity) & leakage.

2) NUCLEIC ACID SYNTHESIS INHIBITORS (E.g., Flucytosine aka 5-fluorocytosine)Used in conjunction with Amphotericin B to treat cryptococcosis or candidiasis.Mode of Action: Converted to 5-fluorouracil (by fungal cytosine deaminase), interfers withactivity of thymidylate synthetase and synthesis of DNA, RNA and proteins.

3) ERGOSTEROL SYNTHESIS INHIBITORS (E.g., Azoles & Allylamines) A. Azoles - Contains 2 groups: Imidazoles (E.g., Ketoconazole) and Triazoles (E.g.,Fluconazole, Voriconazole & Itraconazole).Mode of Action: Block cytochrome P450-dependent 14 α-demethylation of lanosterol(precursor of ergosterol), resulting in inhibiting cell membrane synthesis, accumulation of precursors causes structural and functional characteristic changes of the membrane.

B. Allylamines (E.g., Terbinafine) Broad spectrumMode of Action: Inhibits squalene epioxidase, prevents conversion of squalene → lanosterol,accumulation of squalene increases membrane permeability.N.B: Recognise the stages in ergosterol synthesis (enzymes/structures) (See Fig 70-4, p706,Murray et al.)

4) β-GLUCAN SYNTHESIS INHIBITORS (E.g., Caspofungin, Micafungin & Anidulafungin)Semi-synthetic lipopeptides.Mode of Action: Disrupt cell wall integrity by inhibiting 1,3-β-glucan synthetase

5) MICROTUBULE ASSEMBLY INTERFERENCE (E.g. Griseofulvin)Derived from Penicillium spp. Used to treat DermatophytosesMode of Action: Interacts with microtubules, disrupts mitotic spindle function, results ininhibition of growth (hyphal).

6) UNDER INVESTIGATION• Cell wall synthesis inhibitors (prevent chitin formation) (E.g., Nikkomycin-Z &

Pradimicin)• Elongation factor 2 inhibitors (prevent protein synthesis) (E.g., Sordarin & Azasordarin)

ANTIFUNGAL RESISTANCE (See Table 70-4, p711, Murray et al.) A variety of clinical, cellular and molecular factors can contribute to resistance:• Clinical: Immune status, Site of infection; Severity of infection; Presence of foreignmaterials; Abscess formation; Patient non-compliance!!!!!• Cellular: Cell type (yeast/hyphae); Genomic stability of strain; Phenotypic switching;Size of population; Growth as a biofilm.• Molecular: Alterations in drug import, intracellular drug processing, target enzyme,other enzymes in ergosterol biosynthetic pathway, efflux pumps.

_________________________________________________

Review Questions 1. Why is it important to consider all factors that can influence the effectiveness of a

control agent? 2. Think about medical devices, instruments, patient’s clothing and other objects in a

patient’s hospital room and determine which can be sterilised or disinfected and whatmethod should be used?

3. In addition to knowing the MIC for a specific drug, what other considerations must beaddressed before a drug is prescribed for therapy?



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4. If antibiotics are capable of inhibiting or killing bacteria, explain how bacteriaresponsible for producing some of these antibiotics can survive. E.g., chlortetracyclineis produced by the bacterium Streptomyces aureifaciens. Give several of how thisimmunity to the antibiotic can be accomplished.

5. Explain the attributes of using drugs in combination for infectious disease. List somediseases or conditions which require the use of drugs in combination.

6. Describe the mechanisms by which antibiotic resistance genes on chromosomes as

well as plasmids can be transferred to other chromosomes or genetic units.

_________________________________________________

References in Murray, P.R., Rosenthal, K. S. & Pfaller, M.A. (2009) Medical Microbiology, 6 th Edition (Recommended Course Text)

Chapter 8: Sterilization, Disinfection & Antisepsis p79-83Chapter 20: Antibacterial Agents p199-208Chapter 3: Bacterial Metabolism & Genetics p34-37Chapter 70: Antifungal Agents p701-713

Websites

Mulvey, M.R. & Simor, A.E. (2009) Antimicrobial resistance in hospitals: How concernedshould we be? Canadian Medical Association Journal. 180 (4), p408-415.http://www.cmaj.ca/cgi/content/full/180/4/408?etoc

Hiller, K.M. & Li, J. (2009) Antibiotics: A Review of ED Use. EMedicine from WebMD, Updated16th April 2009 http://emedicine.medscape.com/article/810704-overview viewed on October 20th 2009.

Ghannoum, M.A., & Rice, L.B. (1999) Antifungal Agents: Mode of Action, Mechanisms of Resistance, and Correlation of These Mechanisms with Bacterial Resistance. ClinicalMicrobiology Reviews, Vol. 12, No. 4, p. 501–517. http://cmr.asm.org/cgi/reprint/12/4/501.pdf

McDonnell, G., & Russell, A.D. (1999) Antiseptics & Disinfectants: Activity, Action &Resistance. Clinical Microbiology Reviews, Vol. 12, No. 1, p. 147-179.http://www.ncbi.nlm.nih.gov/pmc/articles/PMC88911/pdf/cm000147.pdf

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