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multiple copies of its specific 16S rRNA gene, thereby increasing the sensitivity of the assay. When

the probe is bound to the target, the label will give off a signal after the free probe is washed away. A

limitation of standard direct probe hybridization is the requirement for a 104

or greater number of

copies of target nucleic acid for detection.

Nucleic acid amplification for diagnosis

Nucleic acid amplification overcomes the principal limitation of direct detection with nucleic acid

probes by selectively amplifying specific DNA targets present in low concentrations. The bacterial

16S rRNA gene has emerged as the most useful marker for microbial detection and identification.

Ribosomal DNA genes contain highly conserved areas (that are used as targets for primers) separated

by internal transcribed sequences containing variable, species-specific regions. These sequences are

like fingerprints. Comparing certain locations on a 16s rRNA gene with a database of known

organisms allows the identification of organisms. For virus detection, primers are constructed to target

highly conserved DNA or RNA sequences unique to the pathogen. Amplification and detection of the

viral genomes are highly sensitive and are especially valuable when the viral load is too low to be

detected by culture or when results are needed rapidly.

Conventional polymerase chain reaction

DNA polymerase repetitively amplifies targeted portions of DNA (ideally sequences that are highly

conserved and unique to the pathogen). Each cycle of amplification doubles the amount of DNA in the

sample, leading to an exponential increase in DNA with repeated cycles of amplification. The

amplified DNA sequence can then be analyzed by gel electrophoresis, Southern blotting, or direct

sequence determination.

Real-time polymerase chain reaction (RT-PCR)

This variant of PCR combines nucleic acid amplification and fluorescent detection of the amplified

product in the same closed automated system. Real-time PCR limits the risk of contamination and

provides a rapid (30-40 minutes) diagnosis. Real-time PCR is a quantitative method and allows the

determination of the concentration of pathogens in various samples.

Advantages of polymerase chain reaction:

1- Methods employing nucleic acid amplification techniques have a major advantage over direct

detection with nucleic acid probes because amplification methods allow specific DNA or RNA

target sequences of the pathogen to be amplified millions of times without having to culture the

microorganism itself for extended periods

2- PCR also permits identification of non-cultivatable or slow-growing organisms, such as

Mycobacteria, anaerobic bacteria, and viruses

3- Nucleic acid amplification methods are sensitive, specific for the target organism, and are

unaffected by the prior administration of antibiotics

4- Nucleic acid amplification techniques are generally quick, easy, and accurate

5- They are useful in the detection of organisms that require complex media or cell cultures or

prolonged incubation times

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PCR amplification is limited by the occurrence of spurious false-positives due to cross-contamination

with other microorganisms’ nucleic acid. PCR tests are often costly and require skilled personnel

DNA microarrays

Although microarrays are now routinely used to measure gene expression, the technique is an

emerging technology in the diagnostic microbiology laboratory. Microarrays have the unprecedented

potential to simultaneously detect and identify many pathogens from the same specimen. For example,

an oligonucleotide microarray targeting the 16S rRNA gene has been developed for the detection of a

panel of forty predominant human intestinal bacterial pathogens in human fecal samples. DNA

microarray consists of microscopic spots of immobilized DNA oligonucleotide, each containing

specific DNA sequences, known as probes. The probes are constructed to be complementary to

specific gene sequences of interest in suspected pathogens. DNA of the microorganism obtained from

a clinical specimen, known as the target, is extracted and amplified using PCR and fluorescent

labeling techniques. The target DNA is exposed to the probe microarray. If the labeled DNA from the

microorganism and the immobilized probe has a complementary base sequence, they will hybridize,

thereby increasing fluorescence intensity. After washing off of nonspecific bonding sequences only

strongly paired strands will remain hybridized and fluoresce. The intensity of fluorescence at each spot

is a measure of the amount of that particular microbial DNA in the sample. Correlating fluorescence

with the identity of the probe allows for the detection and quantitation of specific pathogens.

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Lecture one

Sterilization and Disinfection

Sterilization is the process of killing or removing all viable organisms achieved by: physical and chemical

Disinfection is the process of removing or killing most but not all, viable organisms

Methods:

1. Chemical the substance is called a disinfectant it kills pathogens but may not kill viruses or spores

2. Physical process boiling or low pressure steam it reduces only the bioburden

Antiseptics: A particular group of disinfectants which used to reduce the number of viable organisms in the skin;

that act differentially on organism and host tissue

1. Germicide is a chemical agent capable of killing microbes

2. Sporicide is a germicide capable of killing bacterial spores

Pasteurization: it reduces the total number of viable microbes in bulk fluids such as milk and fruit juices without

destroying flavor and palatability.

Decision whether to use Sterilization or Disinfection it depended upon:

Cost

Damage involved

Low bioburden is a prerequisite for cost-effective sterilization.

Uses of sterilization and disinfection

1. Prevention of hospital infection:

a. sterile equipments, instruments and dressings

b. isolation facilities

c. safe disposal of infected materials

Figure shows the Antiseptics effect on normal skin flora

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2. Microbiologists: production of sterile media and the laboratory activities

3. Central to almost all areas of medical practice like surgery (from hand washing to needles and prosthesis)

Process: The rate of killing of microorganisms depends upon the concentration of the killing agent and time of

exposure.

N = 1/CT

N – Number of survivors

C – Concentration of agent

T – Time of exposure to the agent

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Mechanisms of action of antimicrobial agents:

1. Damage cell membrane

2. Denature proteins

3. Modify functional groups of proteins and nucleic acids

Activity of a particular disinfectant may result from one or combination of pathways.

Factors affecting efficacy:

1. Physical environment

2. Presence of moisture

3. Temperature and pH

4. Concentration of the agent

5. Hardness of water

6. Bioburden and the object

7. Mature and state of microbes in bioburden

8. Ability of microbes to inactivate the chemical agent

Techniques for Sterilization

I. Heat the preferred choice because of:

a. ease of use

b. controllability

c. low cost

d. efficiency

Types:

1. Dry heat

 Incineration

 use of Bunsen burner

 Hot air oven (one hour) 160-180 °C / 1 hour.

2. Moist heat under pressure

a- Autoclave the most effective for sterilization (121 °C / 15 minutes) in which the pressure aids in penetration;

in sterilization of surgical instruments, dressing and heat resistant pharmaceuticals or culture media

b- Boiling water for a few minutes can be used as a rapid emergency measure to disinfect instruments; it kills

vegetative bacteria but not all spores

c- Pasteurization: done at 62.8 – 65.6°C for 30 minutes, which used for fluids as it reduce the number of bacteria

and eliminate pathogen present in small numbers

Flash pasteurization (71.7 °C for 15 seconds). After the process, the fluid should be kept at a temperature below

10°C to minimize, subsequent bacterial growth.

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II. Irradiation: it include gamma and X-rays

Gamma irradiation which used for sterilizing large batches of small volume items such as:

1. Needles, syringes, catheters, gloves

2. Vaccines

3. To prevent food spoilage

4. Capital cost is high but process is 100% efficient

5. Killing mechanism involves production of free radicals that break the bonds in DNA

6. Sporocidal at higher doses (4.5 megarads)

III- Filtration

1-Used to produce particles and pyrogen-free fluid.

2-Composed of nitrocellulose

3-Work by electrostatic attraction and physical pore size

4-Purify drinking water

5-To recover very small number of organism from large volumes of fluid

6-Can be used for quantitating bacteria in fluids

Disinfection by chemical agents

1- Alkylating agents which include gases as

a- ethylene oxide (toxic and explosive)

b-Formaldehyde (extremely unpleasant odor; and irritant to mucous membrane decontaminate rooms and exhaust

protective cabinets)

2- Liquid

I- Glutaraldehyde - disinfect heat–sensitive articles (endoscopes surfaces)

II- Oxidizing agents

1-Hydrogen peroxide (H2O2)

3 – 6% kills most bacteria

10-25% kills all including spores

(Disinfects plastic implants, contact lenses and surgical prosthesis)

III-Halogens it includes:

a-Iodine compounds

1-Most effective, virtually effective among all organisms including spore- formers and Mycobacterium

2-Effective in acid pH because more free iodine is liberated

3-Acts more rapidly than other halogens and quaternary NH4 (ammonium) compounds

4- Effectivity reduced by serum, feces, body fluids.

Povidone iodine used to disinfect metal surfaces and tissues.

b-Chlorine compounds

It available in 3 forms:

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Cl2

HOCl (hypochlorous acid)

OCl (hypochlorite)

It has a good germicide activity, though spores are resistant to it.

IV. Phenolic compounds

a. rarely used as disinfectant nowadays

b. historical interest it was used as comparative standard for assessing the activity of germicidal compounds

V- Quaternary Ammonium compounds which are organic groups linked to nitrogen (Benzalkonium chloride and

Cetyl pyridinium chloride. Bacteriostatic at low concentrations, bactericidal at high. Not effective against

Pseudomonas, Mycobacterium and Trichophyton)

VI-Alcohols

 Germicidal activity increases with increasing chain length (5-8 carbons)

 Most commonly used – ethanol and isopropanol

 No activity against spores

 Activity is more active in the presence of water hence 70% alcohol is more active than 95% alcohol.

 Common disinfectants used for skin surfaces

 Extremely effective when followed by treatment with iodophor

VII- Heavy metals

Soluble salts of Hg (mercury), arsenic, silver and other heavy metals

By forming mercaptides with sulfhydryl groups of cysteine residue

It examples as mercurials – merthiolate, mercurochrome

On the other hand the silver compounds – irritant and caustic effect; silver nitrate (propylaxis); sulfadiazine

cream, colloidal silver compounds used in ophthalmology.

 Antimicrobial agents Chemotherapy: The use of drugs to

treat a disease

 Antimicrobial drugs: Interfere with the growth of microbes within a host

 Antibiotic: It is of biological origin and Produced by a microbe, in order to inhibits other microbes

 Chemotherapeutic agent: synthetic chemicals

 Selective toxicity: Drug kills pathogens without damaging the host

 Therapeutic index: ratio between toxic dose and therapeutic dose or ratio of LD50 (lethal) to ED50

(effective).

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 Antimicrobial action – either Bacteriostatic (which inhibit microbes without destruction) or

bactericidal (destruct microbes and lyses them)

 Some factors effect antibiotics action in our body like:

Tissue distribution, metabolism, and excretion, passage through blood brain barriers (BBB); Unstable in

acid; half-life duration or it shelf life.

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Lecture Two

Selection of antibiotics

Factors to take into consideration:

 The identity of the infecting organism.

 Drug sensitivity of the infecting organism

 Host factors (i.e. site of the infection, status of host defenses).

 Empiric therapy prior to completion of lab tests: it may be necessary to begin treatment in

patients with serious infections BEFORE the lab results.

 Take samples for culture PRIOR TO INITIATION of treatment

HOST FACTORS

 Host defenses (immune system and phagocytic cells).

 Site of infection .To be effective an antibiotic must be present in the site of infection in a

concentration greater than MIC

(Endocarditis, meningitis, abscesses)

 Age (infants and elderly highly vulnerable to drug toxicity).

 Pregnancy and lactation

 Previous allergic reactions

 Genetic factors (i.e. hemolysis in patients with G-6PD deficiency if given sulfonamides).

Antibiotic combinations:

The result may be additive, potentiative or antagonistic.

Additive response: one in which the antimicrobial effect of the combination is equal to the sum of the effects

of the two drugs alone.

Potentiative interaction: one in which the effect of the combination is GREATER than the sum of the

effects of the individual agents.

Antagonistic response: in certain cases the combination of two antibiotics may be less effective than one of

the agents by itself (i.e. combination of a bacteriostatic with a bactericidal drug)

Disadvantages of antibiotic combinations

1) Increased risk of toxic and allergic reactions

2) Possible antagonism of antimicrobial effects

3) Increased risk of suprainfection

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Figure shows the four main Actions of Antimicrobial Drugs

Figure shows the Inhibition of Protein Synthesis by Antibiotics

Mechanism of Action Drugs

 Inhibition of Cell Wall Synthesis

Inhibit cross-linking of peptidoglycan by inactivating transpeptidases (PBPs)

Penicillins, Cephalosporins, Aztreonam, Imipenem

Bind to terminal D-ala-D-ala & prevent incorporation into growing peptidoglycan

Vancomycin, Teicoplanin

Inhibition of transglycosylation

Oritavancin, Teicoplanin, lipophilic vancomycin analogs, ramiplanin

Inhibit dephosphorylation of phospholipid carrier in peptidoglycan structure

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Bacitracin

Prevents incorporation of D-alanine into peptidoglycan

Cycloserine

 Inhibition of Protein Synthesis

Bind to 50S ribosomal subunit

Macrolides, Chloramphenicol, Clindamycin

Bind to 30S ribosomal subunit

Aminoglycosides, Tetracyclines

 Inhibition of Nucleic acid synthesis

Inhibition of DNA gyrase & topoisomerase

Quinolones

Inhibition of nucleic acid biosynthesis

Flucytosine, Griseofulvin

Inhibition of mRNA synthesis

Rifampin, Rifabutin, Rifapentine

 Alteration of Cell Membrane Function

Inhibition of ergosterol biosynthesis

Imidazole antifungals

Bind to membrane sterols

Polymyxins, Amphotericin B, Nystatin

 Alteration of Cell Metabolism

Inhibition of tetrahydrofolic acid production (cofactor for nucleotide synthesis)

Sulfonamides, Trimethoprim, Trimetrexate Pyrimethamine

Inhibition of mycolic acid biosynthesis

Isoniazid

Interference with ubiquinone biosynthesis & cell respiration

Atovaquone

Bind to macromolecules (Metronidazole, Nitrofurantoin)

Antibacterial Antibiotics Inhibitors of Cell Wall Synthesis

Since the gram-positive cell wall contains only two major components it is much less complicated than the

gram-negative cell wall.

Teichoic acids are polymers that are interwoven in the peptidoglycan layer and extend as hair-like

projections beyond the surface of the gram-positive cell. They also are major surface antigens in those

organisms that possess them.

The peptidoglycan layer, or murein layer, of gram-positive bacteria is much thicker than that of gramnegative bacteria. It is responsible for maintaining the shape of the organism and often is referred to as the

cell wall.

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Penicillin is natural and semi synthetic penicillins contain β-lactam ring and natural penicillins produced by

Penicillium are effective against Gram + cocci and spirochetes while semi synthetic penicillins: made in

laboratory by adding different side chains onto β-lactam ring will lead to production of penicillin which are

resistant and broader spectrum of activity

Figure shows The Penicillins nucleus morphology

Figure shows The Penicillins Activity Cycle in the body

Penicillinase (β-lactamase): bacterial enzyme that destroys natural penicillins

Penicillinase resistant penicillins: methicillin replaced by oxacillin and nafcillin due to MRSA

Extended-spectrum penicillin: Ampicillin, amoxicillin; new: carboxypenicilins and ureidopenicillins (also

good against P. aeruginosa)

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Cephalosporin

Produced by fungi of genus Cephalosporium, it Structure and mode of action resembles penicillin, there is 4

Generations of cephalosporin

1. First-generation: Narrow spectrum, gram-positive

2. Second-generation: Extended spectrum includes gram-negative

3. Third-generation: Includes pseudomonads; mostly injected some oral.

4. Fourth-generation: Most extended spectrum

Cephalosporins are:

1. More stable to bacterial lactamase than penicillin

2. Broader spectrum and used against penicillin-resistant strains

Vancomycin

 It is a glycopeptides from Streptomyces

 Inhibition of cell wall synthesis

 Used to kill MRSA

 Emerging Vancomycin resistance: VRE and VRSA

Tetracyclines

A number of antibiotics, including tetracyclines, aminoglycosides, and macrolides, exert antimicrobial

effects by targeting the bacterial ribosome, which has components that differ structurally from those of the

mammalian cytoplasmic ribosomes. Binding of tetracyclines to the 30S subunit of the bacterial ribosome is

believed to block access of the amino acyl-tRNA to the mRNA-ribosome complex at the acceptor site,

thereby inhibiting bacterial protein synthesis.

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Tetracyclines are broad-spectrum antibiotics (that is, many bacteria are sensitive to these drugs.

Tetracyclines are generally bacteriostatic.

Aminoglycosides

Aminoglycosides inhibit bacterial protein synthesis. Susceptible organisms have an oxygen-dependent

system that transports the antibiotic across the cell membrane. All aminoglycosides are bactericidal. They

are effective only against aerobic organisms because anaerobes lack the oxygen-requiring transport system.

Gentamicin is used to treat a variety of infectious diseases including those caused by many of the

Enterobacteriaceae and, in combination with penicillin, endocarditis caused by viridans-group Streptococci.

Macrolides

Macrolides are a group of antibiotics with a macrocyclic lactone structure. Erythromycin was the first of

these to find clinical application, both as the drug of first choice and as an alternative to penicillin in

individuals who are allergic to β-lactam antibiotics. Newer macrolides, such clarithromycin and

azithromycin, offer extended activity against some organisms and less severe adverse reactions.

The macrolides bind irreversibly to a site on the 50S subunit of the bacterial ribosome, thereby inhibiting the

translocation steps of protein synthesis. Generally considered to be bacteriostatic, they may be bactericidal at

higher doses.

Fluoroquinolones

Fluoroquinolones uniquely inhibit the replication of bacterial DNA by interfering with the action of DNA

gyrase (topoisomerase II) during bacterial growth. Binding Quinolones to both the enzyme and DNA to

form a ternary complex inhibits the rejoining step, and, thus, can cause cell death by inducing cleavage of

the DNA.

Because DNA gyrase is a distinct target for antimicrobial therapy, cross-resistance with other more

commonly used antimicrobial drugs is rare but is increasing with multidrug-resistant organisms.

All of the Fluoroquinolones are bactericidal.

Carbapenems

Carbapenems are synthetic β-lactam antibiotics that differ in structure from the penicillins. Imipenem,

meropenem, doripenem, and ertapenem are the drugs of this group currently available. Imipenem is

compounded with cilastatin to protect it from metabolism by renal dehydropeptidase. Imipenem resists

hydrolysis by most β-lactamases.