THE CONTROL OF THE CONTROL OF MICROORGANISMS BY MICROORGANISMS BY

CHEMICAL AND PHYSICAL CHEMICAL AND PHYSICAL FACTORS FACTORS

EXERCISE 18. THE ANTIBIOTIC SENSITIVITY TESTING

EXERCISE 19. THE FILTER PAPER DISK METHOD:

A. EVALUATION OF DISINFECTANTS AND ANTISEPTICS B. THE INHIBITORY ACTION OF HEAVY METALS AND OTHER

CHEMICALS

Microbial control by chemical and physical

“the use of antiseptics, disinfectants, antibiotics, ultraviolet light (UV) and many other agents”

The process of destroying all forms of life. A sterile object is one free of all life forms, including endospores. The reduction or elimination of pathogenic microorganisms in or on materials, so they are no longer a health hazard. Chemical agents used to disinfect inanimate objects but generally to toxic to use on human tissues.

Chemical agents that disinfect, but are not harmful to human tissues. A metabolic product produced by one microorganism that inhibits or kills other microorganisms.

Synthetic chemicals that can be used therapeutically.

Kills microorganisms.

Inhibits the growth of microorganisms.

1. Sterilization:

2. Disinfection:

3. Disinfectant:

4. Antiseptic:

5. Antibiotic:

6. Chemotherapeutic antimicrobial chemical:

7. Cidal:

8. Static:

Basic terms used in discussing the control of microorganisms

1.the concentration and kind of a chemical agent used 2. the intensity and nature of a physical agent used

3. the length of exposure to the agent 4. the temperature at which the agent is used

5. the number of microorganisms present

6. the organism itself

7. the nature of the material bearing the microorganism.

When evaluating or choosing a technique to control microorgansims there are some factors which may influence antimicrobial activity:

EXERCISE 18: THE ANTIBIOTIC SENSITIVITY TESTING

ANTIBIOTICs (anti: against; bios: life)

“metabolic by-products of one microorganism that is able to kill or inhibit other microorganisms”

Alexander Fleming and the crude antimicrobial extract obtained from Penicillium notatum was named as PENICILLIN

“FIRST DISCOVERED ANTIBIOTIC”

BACTERICIDAL “physical and chemical agent that is able to kill some types of bacteria”(e.g., penicillins, cephalosporins, streptomycin)neomycin)

BACTERIOSTATIC“any agent that inhibits the growth and reproduction of some types of bacteria but need not kill the bacteria”(e.g., tetracyclines, erythromycin, sulfonamides)

1. antibiotics: substances produced as metabolic products of one microorganism which inhibit or kill other microorganisms. 2. antimicrobial chemotherapeutic chemicals: chemicals synthesized in the laboratory which can be used therapeutically on microorganisms.

Today the distinction between the two classes is not as clear, since many antibiotics are extensively modified in the laboratory (semisynthetic) or even synthesized without the help of microorganisms.

Based on their origin, there are 2 general classes of antimicrobial chemotherapeutic agents:

Antimicrobial agents also vary in their spectrum..

BROAD SPECTRUM

tetracycline, streptomycin, cephalosporins,ampicillin, sulfonamides

NARROW SPECTRUM

penicillin G,erythromycin, clindamycin,gentamicin

Drugs that are effectiveagainst a variety of both gram-positive andgram-negative bacteria

effective against justgram-positive bacteria, just gram negative bacteria,or only a few species

a narrow spectrum is preferable cause less destruction of body’s normal flora prevents superinfection by opportunistic microorganisms, drug toxicity, allergic rxns, selection of resistant m/o

The antimicrobial activity of an antibiotic can be tested by several methods designed to determine the smallest amount of the agent needed to inhibit the growth of a microorganism.

Commonly used methods to determine MIC and MBC are “agar diffusion method” also known as Kirby-Bauer method and “dilution method”.

Minimal Inhibitory Concentration (MIC) The smallest amount of the agent needed to inhibit the growth of

a microorganism

MMinimal BBactericidal CConcentration (MBC)the minimum amount of agent required to kill the microorganism

Antimicrobials are usually regarded as bactericidal if the MBC is no more than four times the MIC

Agar diffusion method

a petri plate containing a suitable medium is heavily inoculated by spread plate technique with the microorganism whose antibiotic sensitivity is to be determined

commercially available filter paper disks, each containing defined concentrations of a specific antibiotic, are removed from individual containers and are placed onto the agar surface

during the incubation antibiotics diffuse from the disk into the agar

at some particular distance from each disk, the MIC for the antibiotic is reached and microbial growth is inhibited

the MIC’s are recognized by the presence of “GROWTH INHIBITION ZONES” (clear zones) surrounding the various antibiotic disks used

Escherichia coli exhibits varying sensitivity to antibiotics. Of the antibiotics used, this particular strain of E. coli is most sensitive to CTX 100 (Cefotaxime), showing no growth in the zone surrounding the disk

Agar diffusion method

growth inhibition zones” are observed and diameters of such zones are measured milimetrically. The results constitute an ANTIBIOGRAMANTIBIOGRAM.

The relative diameters of the zones do not necessarily indicate the relative activities of the chemotherapeutic agents used in the test. The size of the growth inhibition zones can be affected by several factors including:

1. the culture medium used 2. incubation conditions (temperature, oxygen availability, time period and etc.) 3. the rate of diffusion of antibiotic 4. the concentrations of the antibiotics used 5. antibiotic sensitivity of the organism being tested.

Agar diffusion method (cont’d)

The Kirby-Bauer test (agar diffusion method) must be carefully standardized.

•Special agar, i.e. Mueller-Hinton agar, is used along with a prescribed inoculum of broth. •The antibiotic disks are also standardized to contain a specific amount of antibiotic. •A specified period of time of incubation at specified temperature,

“the clear zones” are measured.

These are compared with tables giving the interpretation of measurement for each antibiotic.

This unsupplemented medium has been selected by the National Committee for Clinical Laboratory Standards (NCCLS)1 for several reasons:5 this medium is low in sulfonamide, trimethoprim and tetracycline inhibitors, provides satisfactory growth of most non-fastidious pathogens and demonstrates batch-to-batch reproducibility. Mueller Hinton Agar is often abbreviated as M-H Agar, and complies with requirements of the World Health Organization.5 Mueller Hinton Agar is specified in FDA Bacteriological Analytical Manual6 for food testing, and procedures commonly performed on aerobic and facultatively anaerobic bacteria.7

A variety of supplements can be added to Mueller Hinton Agar, including 5% defibrinated sheep or horse blood, 1% growth supplement and 2% sodium chloride. Principles of the Procedure Beef Extract and Acid Hydrolysate of Casein provide nitrogen, vitamins, carbon, and amino acids in Mueller Hinton Agar. Starch is added to absorb any toxic metabolites produced. Agar is the solidifying agent. A suitable medium is essential for testing the susceptibility of microorganisms to sulfonamides and trimethoprim. Antagonism to sulfonamide activity is demonstrated by para-aminobenzoic acid (PABA) and its analogs. Reduced activity of trimethoprim, resulting in smaller growth inhibition zones and inner zonal growth, is demonstrated on medium possessing high levels of thymide. The PABA and thymine/thymidine content of Mueller Hinton Agar are reduced to a minimum, reducing the inactivation of sulfonamides and trimethoprim. Formula / Liter Beef Extract...............................................................................2 g Acid Hydrolysate of Casein..................................................17.5 g Starch.....................................................................................1.5 g Agar.........................................................................................17 g Final pH 7.3 ± 0.1 at 25°C Formula may be adjusted and/or supplemented as required to meet performance specifications.

Mueller Hinton Agar

Eukaryotic microorganisms, on the other hand, have structures and functions more closely related to those of the host. As a result, the variety of agents selectively effective against eukaryotic microorganisms such as fungi and protozoans is small when compared to the number available against prokaryotes.

Viruses are not cells and, therefore, lack the structures and functions altered by antibiotics so antibiotics are not effective against viruses.

Antimicrobial chemotherapy

SELECTIVE TOXICITY WITHOUT seriously harming the host

In order to be selectively toxic, a chemotherapeutic agent must interact with some microbial function or microbial structure that is either not present or is substantially different from that of the host.

In treating infections caused by prokaryotic bacteria, the agent may inhibit peptidoglycan synthesis or alter bacterial (prokaryotic) ribosomes. Human cells do not contain peptidoglycan and possess eukaryotic ribosomes. Therefore, the drug shows little if any, effect on the host

Commonly used antimicrobial chemotherapeutic agents

arranged according to their mode of action:

Inhibitpeptidoglycan synthesis

alter the cytoplasmic membrane

InhibitProteinsynthesis

Interferewith DNAsynthesis

Principle of antibiotic spectrumPrinciple of antibiotic spectrum

Different antibiotics target different kinds of bacteria◦ i.e., different spectrum of activity

Examples:◦ Penicillin G (= original pen.) mainly streptococci (narrow

spectrum)◦ Vancomycin only Gram-positive bacteria (intermediate

spectrum)◦ Carbapenems many different bacteria (very broad

spectrum)

1. 1. Antimicrobials acting on Antimicrobials acting on the bacterial cell wallthe bacterial cell wall

Interfere with synthesis of peptidoglycan layer in cell wall of actively dividing bacteria◦ eventually cause cell lysis◦ bind to and inhibit activity

of enzymes responsible for peptidoglycan synthesis “penicillin-binding

proteins”

Antimicrobials acting on Antimicrobials acting on the bacterial cell wallthe bacterial cell wall

Beta-lactams: Penicillins◦benzylpenicillin ◦flucloxacillin ◦ampicillin ◦piperacillin B-lactam ring

Beta-lactams: Cephalosporins◦ Orally active

cephradine cephalexin

◦ Broad spectrum cefuroxime cefotaxme ceftriaxone ceftazidime

Cephalosporins

synthetic side chains change the spectrum

of action

a house with a garage & basement

Antimicrobials acting on Antimicrobials acting on the bacterial cell wallthe bacterial cell wall

Unusual beta-lactams◦ Carbapenems

Imipenem, meropenem◦ very wide spectrum

◦ Monobactams Aztreonam

◦ only Gram-negatives Glycopeptides

◦ only Gram-positives, but broad spectrum

◦ vancomycin◦ teicoplanin

Antimicrobials acting on Antimicrobials acting on the bacterial cell wallthe bacterial cell wall

Antimicrobials acting on Antimicrobials acting on nucleic acid synthesis nucleic acid synthesis

Inhibitors Of Precursor Synthesis◦ sulphonamides & trimethoprim are synthetic,

bacteriostatic agents used in combination in co-trimoxazole Both of these drugs block enzymes in the bacteria pathway

required for the synthesis of tetrahydrofolic acid, a cofactor needed for bacteria to make the nucleotide bases thymine, guanine, uracil, and adenine.

◦ Sulphonamides inhibit early stages of folate synthesis dapsone, an anti-leprosy drug, acts this way too

◦ Trimethoprim inhibits final enzyme in pathway, dihydrofolate synthetase. pyramethamine, an anti-toxoplasma and anti-PCP drug acts

this way too

Inhibitors of DNA replication ◦ Quinolones (e.g ciprofloacin) inhibit DNA-gyrase◦ Orally active, broad spectrum

Damage to DNA◦ Metronidazole (anti-anaerobes), nitrofurantoin (UTI)

Inhibitors of Transcription ◦ rifampicin (key anti-TB drug) inhibits bacterial RNA

polymerase◦ flucytosine is incorporated into yeast mRNA

2. 2. Antimicrobials acting on Antimicrobials acting on nucleic acid synthesis nucleic acid synthesis

3. 3. Antimicrobials acting on Antimicrobials acting on protein synthesis protein synthesis

Binding to 30s Subunit◦ aminoglycosides (bacteriocidal)

streptomycin, gentamicin, amikacin.◦ tetracyclines

Binding to the 50s subunit◦ chloramphenicol ◦ fusidic acid ◦ macrolides (erythromycin, ◦ clarithromycin, azithromycin)

◦ Agents that block TRANSCRIPTION◦ i.e. rifampisin◦ ◦ Agents that block TRANSLATION

30s subunit

50s subunit

mRNA

protein

These agents prevent bacteria from synthesizing structural proteins and enzymes

4. 4. Antimicrobials acting on Antimicrobials acting on the cell membranethe cell membrane

Alteration of the cytoplasmic membrane of microorganisms results in leakage of cellular materials

amphotericin binds to the sterol-containing membranes of fungi

polymyxins act like detergents and disrupt the Gram negative outer membrane.◦ Not used parenterally because of toxicity to mammalian cell

membrane

fluconazole and itraconazole interfere with the biosynthesis of sterol in fungi

Mechanisms of resistanceMechanisms of resistance on on microorganisms against antibioticsmicroorganisms against antibiotics1. Producing enzymes which detoxify or inactivate the antibiotic, e.g., penicillinase and other beta-lactamases.

2. Altering the target site in the bacterium to reduce or block binding of the antibiotic, e.g., producing a slightly altered ribosomal subunit that still functions but to which the drug can't bind.

3. Preventing transport of the antimicrobial agent into the bacterium (reducing permeability of membranes), eg., producing an altered cytoplasmic membrane or outer membrane.

4. Developing an alternate metabolic pathway that is not affected by the drug; to by-pass the metabolic step being blocked by the antimicrobial agent,

5. Increasing the production of the target,

6. Efflux of antimicrobial agent.

Resistance can arise from chromosomal mutations, or from acquisition of resistance genes on mobile genetic elements (R plasmids mainly found in gram - bacteria◦ plasmids, transposons, integrons

Resistance determinants can spread from one bacterial species to another, across large taxonomic distances

Multiple resistance determinants can be carried by the same mobile element◦ Tend to stack up on plasmids

Mechanisms of resistanceMechanisms of resistance on on microorganisms against antibiotics microorganisms against antibiotics (cont’d)(cont’d)

Spread of Antimicrobial Drug Spread of Antimicrobial Drug ResistanceResistance

Inappropriate, extensive use of antimicrobial drugs is leading to the rapid development of drug-resistance in disease-causing microorganisms.

Drugs prescribed for treatment of a particular infection have changed because of increased resistance of the microorganism causing the disease.

Ex. Neisseria gonorrhoeae is now resistant to penicillin. Antibiotic treatment is warranted in 20% of individuals who

are seen for clinical infectious disease, yet antibiotics are prescribed up to 80% of the time. In up to 50% of cases recommended doses or duration of treatments are not correct.

This is compounded by patient noncompliance – how?

Spread of Antimicrobial Drug Spread of Antimicrobial Drug Resistance (cont.)Resistance (cont.)

Other indiscriminant, nonessential uses of antibiotics contribute to the emergence of resistant strains, ex. antibiotics are used in agriculture both a growth-promoting substances in animal feeds and as prophylactics – what does that mean?

If the use of a particular antibiotic is stopped, resistance to that antibiotic may be reversed over time.

Impact of antibiotic resistanceImpact of antibiotic resistance

Infections that used to be treatable with standard antibiotics now need revised, complex regimens:◦ e.g., penicillin-resistant Strep. pneumoniae now requires

broad-spectrum cephalosporin In some instances, hardly any antibiotics left:

◦ e.g., Multiresistant Pseudomonas aeruginosa◦ e.g., Vancomycin-resistant Staph. aureus

Resistance rates worldwide increasing

The Search for New Antimicrobial The Search for New Antimicrobial DrugsDrugs

The production of new analogs of existing antimicrobial compounds is often productive since they mimic the original drugs to some extent and have a predictable mechanism of action, but may be different enough to act on organisms resistant to the original form of the drug.

Application of automated robotic chemistry methods to drug Application of automated robotic chemistry methods to drug discovery discovery = combinatorial chemistry.

Many different derivatives of an antimicrobial agent can be generated in a short time, ex. 725 different tetracycline derivatives from only 6 different reagents in only a few hours.

According to the pharmaceutical industry, ~7 million candidate compounds must be screened to yield a single useful clinical drug. New drug discovery: 10-25 years and $500 million for each new drug approved (FDA).

Computerized drug design can facilitate the design of completely new drugs, ex. a protease inhibitor currently used to treat HIV.

Antibiotic susceptibility testing in the laboratory

EXERCISE 19. THE FILTER PAPER DISK METHOD:

A.EVALUATION OF DISINFECTANTS AND ANTISEPTICS

B. THE INHIBITORY ACTION OF HEAVY METALS AND OTHER CHEMICALS

DISINFECTION is the reduction or elimination of pathogenic microorganisms in or on materials so that they are less of a health hazard. The term DISINFECTANT is generally used for chemical agents employed to disinfect inanimate objects, whereas the term ANTISEPTIC is used to indicate a nontoxic disinfectant suitable for use on animal tissue. Because disinfectants and antiseptics often work slowly on some viruses (such as the hepatitis viruses), Mycobacterium tuberculosis, and especially bacterial endospores, they are usually unreliable for sterilization (the destruction of all life forms).

Originally the term antiseptic was applied to any agent that prevents sepsis, or putrefaction. Since sepsis is caused by growing microorganisms, it follows that an antiseptic inhibits microbial multiplication without necessarily killing them. By this

definition, we can assume that antiseptics are essentially bacteriostatic agents.

1.The concentration of the chemical agent.

2. The temperature at which the agent is being used. Generally, the lower the temperature, the lower the effectiveness.

3. The kinds of microorganisms present (endospore producers, Mycobacterium tuberculosis, etc.).

4. The number of microorganisms present. The more organisms present, the harder it is to disinfect.

5. The nature of the material bearing the microorganisms. Organic material such as dirt and excreta interferes with some agents.

There are a number of factors which influence the antimicrobial action of disinfectants and antiseptics, including:

The best results are generally obtained when the initial microbial numbers are low and when the surface to be disinfected is clean and free of possible interfering substances.

There are 2 common antimicrobial modes of action for disinfectants and antiseptics:

1.They may damage the lipids and/or proteins of the semipermeable cytoplasmic membrane of microorganisms resulting in leakage of cellular materials needed to sustain life.

2. They may denature microbial enzymes and other proteins, usually by disrupting the hydrogen and disulfide bonds that give the protein its three-dimensional functional shape. This blocks metabolism.

Some of the more commonly used disinfectant groups are listed below.

1. Phenol and phenol derivativesThese agents kill most bacteria, most fungi, and some viruses, but are usually ineffective

against endospores. They alter membrane permeability and denature proteins.

2. Soaps and detergents Detergents may be anionic or cationic. Anionic (negatively charged) detergents, such as

laundry powders, mechanically remove microorganisms and other materials but are not very microbicidal. Cationic (positively charged) detergents alter membrane permeability and denature proteins. They are effective against many vegetative bacteria, some fungi, and some viruses. However, endospores, Mycobacterium tuberculosis, and Pseudomonas species are usually resistant. They are also inactivated by soaps and organic materials like excreta. Cationic detergents include the quaternary ammonium compounds (zephiran, diaprene, roccal, ceepryn, and phemerol).

3. Alcohols 70% solutions of ethyl or isopropyl alcohol are effective in killing vegetative bacteria,

enveloped viruses, and fungi. However, they are usually ineffective against endospores and non-enveloped viruses. Once they evaporate, their cidal activity will cease. Alcohols denature membranes and are often combined with other disinfectants, such as iodine, mercurials, and cationic detergents for increased effectiveness.

4. Acids and alkalies Acids and alkalies alter membrane permeability and denature proteins and other molecules.

Salts of organic acids, such as calcium propionate, potassium sorbate, and methylparaben, are commonly used as food preservatives. Undecylenic acid (Desenex®)

is used for dermatophyte infections of the skin. An example of an alkali is lye (sodium hydroxide).

5. Heavy metals Mercury compounds (mercurochrome, metaphen, merthiolate) are only bacteriostatic and are

not effective against endospores. Silver nitrate (1%) is sometimes put in the eyes of newborns to prevent gonococcal ophthalmia. Copper sulfate is used to combat fungal diseases of plants and is also a common algicide. Selinium sulfide kills fungi and their spores.

6. Chlorine Chlorine gas reacts with water to form hypochlorite ions, which in turn denature microbial

enzymes. Chlorine is used in the chlorination of drinking water, swimming pools, and sewage. Sodium hypochlorite is the active agent in household bleach. Calcium hypochlorite, sodium hypochlorite, and chloramines (chlorine plus ammonia) are used to sanitize glassware, eating utensils, dairy and food processing equipment, and hemodialysis systems

Some of the more commonly used disinfectant groups are listed below (cont’d)

7. Iodine and iodophoresIodine also denatures microbial proteins and is usually dissolved in an alcohol solution to

produce a tincture. Iodophores are a combination of iodine and an anionic detergent (such as polyvinylpyrrolidone) which reduces surface tension and slowly releases the iodine. Iodophores are less irritating than iodine and do not stain. They are generally effective against vegetative bacteria, Mycobacterium tuberculosis, fungi, some viruses, and some endospores. Examples include Wescodyne®, Ioprep®, Ioclide®, Betadine®, and Isodine®

8. Aldehydes Aldehydes, such as formaldehyde and glutaraldehyde, denature microbial proteins. Formalin

(37% aqueous solution of formaldehyde gas) is extremely active and kills most forms of microbial life. It is used in embalming, preserving biological specimens, and in preparing vaccines. Alkaline glutaraldehyde (Cidex®), acid glutaraldehyde (Sonacide®), and glutaraldehyde phenate solutions (Sporocidin®) kill vegetative bacteria in 10-30 minutes and endospores in about 4 hours. A 10 hour exposure to a 2% glutaraldehyde solution can be used for cold sterilization of materials.

9. Ethylene oxide gas Ethylene oxide is one of the very few chemicals that can be relied upon for sterilization (after 4-12 hours

exposure). Since it is explosive, it is usually mixed with inert gases such as freon or carbon dioxide. Gaseous chemosterilizers, using ethylene oxide, are commonly used to sterilize heat-sensitive items such as plastic syringes, petri plates,

textiles, sutures, artificial heart valves, heart-lung machines, and mattresses. Ethylene oxide has very high penetrating power and denatures microbial proteins. Vapors are toxic to the skin, eyes, and mucous membranes and are also carcinogenic.

Some of the more commonly used disinfectant groups are listed below (cont’d)

B. THE INHIBITORY ACTION OF HEAVY METALS AND OTHER CHEMICALS

OLIGODYNAMIC ACTION

Heavy metals such as silver, copper and mercury, either alone or in certain compounds have long been known to produce harmful effects on microorganisms..The ability of extremely small quantities of certain metals toexert toxic effects on microorganisms

Demonstration of the oligodynamic action and individual reactions of selected microorganisms to the kinds and concentrations of metals or heavy metal ions again using the

“FILTER PAPER DISK METHOD”

Oligodynamically active metals are used to control microorganisms in a variety of circumstances. Such applications include the treatment of various alcoholic beverages, milk and water, the preparation of antiseptic agents and the impregnation of various fabric

If we are to compare antiseptics on the basis of their bacteriostatic properties, the “FILTER PAPER DISK METHOD” is a simple satisfactory method to use. In this method, a disk of filter paper is impregnated with chemical agent to be tested and placed on a seeded nutrient agar plate. The plate is incubated for a specified period and if the substance is inhibitory, a clear zone of inhibition will surround the disk. The size of this zone is an expression of the agent’s effectiveness and can be compared quantitatively against other substances.

“FILTER PAPER DISK METHOD”