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MICROBIAL GENETICS INTRODUCTION TO GENETICS AND GENES: UNLOCKING THE SECRETS OF HEREDITY Genetics is the study of the inheritance or heredity of living things. Organismic genetics studies the heredity of the whole organism; chromosomal genetics studies the characteristics and actions of chromosomes, and molecular genetics studies the biochemistry of the genes. THE NATURE OF THE GENETIC MATERIAL The Levels of Structure and Function of the Genome: A genome is a complete set of genetic material (genes) in a cell. For example, an ovum or a sperm contains a genome. The genetic material of living things is predominantly DNA. However, the genetic material of some viruses such as HIV and Ebola is RNA. A gene can be defined as a sequence of DNA which codes for the synthesis of one polypeptide. Bacterial chromosome consists of a single molecule of double-stranded deoxyribonucleic acid (DNA) in ring shape which is in association with histonelike proteins. A bacterium contains one chromosome. It contains hereditary information which is passed from one generation to the next generation. The procaryotic chromosome is not surrounded by a nuclear membrane. A bacterium may contain one or more extra piece of chromosomes called plasmids. Plasmids are circular, double-stranded DNA. Linear plasmids have also been found in a few species of bacteria. Plasmids may contain genes responsible for antibiotic resistance. They have been used as vectors to transfer the foreign genes into the bacterial cells in genetic engineering techniques. The Size and Packaging of Genomes: The smallest viruses contain 4 or 5 genes. Escherichia coli contains about 4,000 genes and human cell contains about 100,000 genes. The DNA of E. coli if unwound and stretched out linearly, it measures about 1 mm. If all the DNA in 46 chromosomes in a human cell are linked together to form a linear DNA, it will measure about 6 feet. If all the DNAs in a human body (50 trillion cells) are linked together, its length is more than enough to cover a distance to and back from the moon. A book with one million pages will be made if all the bases (A, T, C, G) found in 46 chromosomes in a human cell are printed. THE DNA CODE: A SIMPLE YET PROFOUND MESSAGE DNA consists of building blocks called nucleotides. A nucleotide consists of a phosphate, a sugar (deoxyribose) and a nitrogenous base (adenine, guanine, cytosine and thymine). Nucleotides link together to form a polynucleotide (strand).. A DNA consists of two strands arranged in an anti-paralleled direction. If one strand goes from the 3' to 5' direction, the other strand always goes in the 5' to 3' direction. These two strands are held together by hydrogen bonds between their nitrogenous bases according to the base-pairing rules: adenine with thymine, and cytosine with guanine. The number of hydrogen bonds between adenine and thymine is 2, and that between cytosine and guanine is 3. THE SIGNIFICANCE OF DNA STRUCTURE The nitrogenous bases influence DNA in two major ways:

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1.

Maintenance of the code during reproduction: During cell division, the DNA strands separate, each serves as a template for the synthesis of a new strand. In this manner, each daughter cell will inherit the identical DNA.

2.

Providing variety: The order of bases along the DNA strands is unique in each organism. A gene is a specific sequence of bases. An endless number of sequences can be made from four nitrogenous bases.

DNA REPLICATION: PRESERVING THE CODE AND PASSING IT ON DNA replication is the process in which DNA is duplicated. In E. coli, the process takes about 20 minutes. The Overall Replication Process: The process involves the following basic steps: (1) uncoiling of DNA, (2) unzipping of the hydrogen bonds so each strand serves as a template for the synthesis of a new strand, and (3) synthesis of a new strand whose bases are complementary to those of the old strand. DNA synthesis is described as semi-conservative type because each old strand is used as a template for the synthesis of a new strand. So, in every DNA molecule one strand is always an old one and another strand is always a new one. Refinements and Details of Replication: The circular bacterial DNA replicates by a special configuration called a replicon (Fig. 9.7, p. 264). During bacterial DNA replication, the circular DNA double helix unwinds due to the action of the enzyme DNA gyrase at a specific site. Replicons are found only in prokaryotic DNA, but not in eukaryotic DNA. The hydrogen bonds are broken by the enzyme DNA helicase ('unzipping enzyme"). (Some books also describe that DNA helicase can also unwind DNA.) The helix separates, forming two replication forks (Fig. 9.7 & 9.8, p. 264-265). The forks move in opposite directions around the circular DNA until they meet. As the DNA strands separate, each strand is used as a template to synthesize a new strand. Synthesis of new DNA strands can occur in only one direction, from the 5' phosphate end to the 3' hydroxyl end of DNA. That is, the nucleotides have to be added from the old 3' hydroxyl end to the 5' phosphate end of DNA. Two new strands are assembled in an anti-parallel direction. The assembly of nucleotides on the old 3'-5' strand is straight forward. It is accomplished by DNA polymerase III. On the old 5'-3' strand, the assembly is discontinuous, forming segments of new nucleotides. Each segment always starts with a RNA primer of about 10 bases. It is synthesized by RNA polymerase. A primer provides a point to which true DNA nucleotides may be added by DNA polymerase III. After a few DNA nucleotides have been connected to the RNA primer by DNA polymerase III, the primer is erased (degraded) by DNA polymerase I (eraser enzyme). The bases which have been erased are replaced with true DNA nucleotides by DNA polymerase II (repair enzyme). (Some books also describe that DNA polymerase I can also remove RNA primer and repair it. This book also says that DNA polymerase III can remove the RNA primers.) DNA fragments, called Okazaki fragments which consist of about 1,000 to 2,000 bases, are formed along the old 5'-3' strand. Many Okazaki fragments are formed in this manner. Each fragment always starts with a RNA primer. These Okazaki fragments are then joined together by DNA ligase (also called polynucleotide ligase or joining enzyme) to form a new polynucleotide. This seemingly wasteful process may serve as a fail-safe procedure of DNA copying, since most errors occur at the beginning of the old copy where the 3' end has not yet been exposed. Elongation and Termination of the Daughter Molecules: The replicon looks like a half-open eye as one duplicating strand droops down (theta 0) (Fig. 9.8, p. 260). When two replication forks come full circle and meet, two DNA molecules are produced.

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The rate of synthesis is about 500 (750 in some bacteria) nucleotides per second in procaryotes and about 50 nucleotides per second in eucaryotes. Other Variations on the Theme: DNA replication in eucaryotes starts at many sites along the DNA. Each site forms a replication bubble. Hundreds of these bubbles eventually fuse when two linear double-stranded DNA molecules are formed. A type of replication called rolling circle occurs in bacteriophages and plasmids (Fig. 9.9, p 265). A new strand is made from the parent DNA as it rotates. A complementary strand is then made from the new strand to form a double-stranded circular DNA. DNA replication is precise. However, occasionally mistakes are made once in about 100,000 to 1 million bases. They are repaired by DNA polymerase III. If mistakes are not repaired, the cell will undergo mutation. APPLICATIONS OF THE DNA CODE: TRANSCRIPTION AND TRANSLATION The central dogma (theme) of molecular biology is the concept that genetic information flows from DNA to RNA to protein. The synthesis of RNA from DNA is called transcription, and the synthesis of protein from RNA is called translation. Exceptions to this concept are found in some RNA viruses which convert RNA to other RNA, and in retroviruses which convert RNA to DNA. Transcription in eukaryotic cells occurs in the nucleus, while translation occurs on ribosomes. THE GENE-PROTEIN CONNECTION There are three basic categories of genes: structural genes code for proteins, genes that code for RNA, and regulatory genes control gene expression. The genotype is the genetic makeup of an organism, and the phenotype is the observable characteristics of an organism (i.e. shape of cell). The Triplet Code and the Relationship to Proteins: The three adjacent bases on one DNA strand constitute a triplet. When a DNA sequence is transcribed into a sequence of RNA, each triplet is called a codon. A codon codes for one amino acid. The uniqueness of an organism is due to the uniqueness of its DNA sequence which dictates the specific order of amino acids in a polypeptide. The relationship between DNA and protein functions are: (1) the primary structure of a protein determines its characteristic shape and function, (2) proteins determine phenotype, and (3) DNA is a blueprint of life. It tells a cell what proteins to make. THE MAJOR PARTICIPANTS IN TRANSCRIPTION AND TRANSLATION RNAs: Tools in the Cell's Assembly Line RNA is a single-stranded molecule (it may also form a hairpin loop, a secondary structure, or a tertiary structure). RNA contains uracil (in place of thymine found in DNA) and ribose (in place of deoxyribose found in DNA). All types of RNA are formed from DNA. Only mRNA can be translated into protein. Messenger RNA: Carrying DNA's Message

Messenger RNA (mRNA) is a transcript (copy) of structural gene or genes complementary to DNA. The triplets (three adjacent bases) in mRNA are called codons. The average length of a mRNA is about 1,500 bases.

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Transfer RNA: The Key to Translation Transfer RNA (tRNA) is a small molecule, consisting of about (85 to 97 bases). When it connects to an amino acid, it transforms into a cloverleaf structure (Fig. 9.12, p. 268). The three unpaired bases constitute an anticodon which has to be complementary to the codon in mRNA in order for the tRNA to deliver an amino acid during translation. Each tRNA is specialized to deliver one amino acid. There are at least 20 different tRNA. The Ribosome: A Mobile Molecular Factory for Translation The procaryotic ribosome (70S) consists of rRNA and protein. It has two subunits: the smaller subunit and the larger subunit. Ribosomes contribute both enzymatic and attachment functions for mRNA and tRNA. A bacterial cell may contain up to 20,000 ribosomes. TRANSCRIPTION: THE FIRST STAGE OF GENE EXPRESSION Only one strand of DNA is transcribed into mRNA. The strand that contains base sequence for synthesis of protein is called the sense strand (template strand). The strand that does not contain information for the synthesis of protein is called the nonsense, noncoding (your textbook is wrong in calling it coding) or antisense strand (Fig. 9.13, p. 270). PROTEIN SYNTHESIS Transcription Translation DNA ------------------ > RNA ----------------- > Protein The segment of DNA that contains the information for the synthesis of a protein is called a gene. Transcription: The process of synthesizing RNA based on the sequence of nucleotides in DNA is called transcription. It involves three phases: initiation, elongation and termination. Initiation: The initiation of transcription begins with the attachment of RNA polymerase on specific locations (sequences) on DNA molecule called promoters. The promoters determine which DNA strand (sense strand) will be transcribed. The enzyme contains a subunit called the sigma factor (a protein). The sigma factor determines which promoter site on DNA will begin transcription. Once the core of RNA polymerase attaches to the promoter site, the sigma factor dissociates from the enzyme. In most cases, the triplet on DNA that begins transcription is TAC which is transcribed as AUG (codes for methionine) on mRNA. Elongation: The segment of DNA that contains genetic information for a specific protein is uncoupled by RNA polymerase. Free ribonucleotides (ATP, UTP, CTP and GTP) are automatically attracted by the exposed bases on one polynucleotide strand (the sense strand) which contains the genetic information. New nucleotides are linked to the exposed bases by hydrogen bonds according to the base-pairing rules: adenine pairs with uracil, A-U; thymine pairs with adenine, T-A; and cytosine pairs with guanine, C-G. The RNA polymerase catalyzes condensation reaction between the newly attached nucleotides by the elimination of inorganic pyrophosphate, forming a new polyribonucleotide which is called messenger-RNA (mRNA). The direction of RNA synthesis is similar to that of DNA, starting from the 5' end to the 3' end of the mRNA. As the mRNA is still being synthesized, its 5' end detaches from the DNA and immediately attaches to the ribosome to initiate translation. In eucaryotes, the mRNA has to move out the nucleus through nuclear membrane before translation can begin. When the mRNA breaks away from the DNA, the two polynucleotide strands of DNA rejoin together.

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Termination: A specific molecule called the rho protein moves along the sense strand. When it encounters the termination sequence, it signals to the RNA polymerase to end the transcription process. The mRNA then breaks away from the DNA. TRANSLATION: THE SECOND STAGE OF GENE EXPRESSION The four phases of translation are: amino acid activation, initiation, elongation and termination. The Beginning Stages of Translation: Initiation Amino Acid Activation: In order for an amino acid to attach to a tRNA, it has to react with ATP first with the action of aminoacyl synthetase to form adenylo-amino acid. It then attaches to the 3' end of tRNA to form a charged tRNA. Once the tRNA picks up an amino acid, it transforms into a clover-leaf shape molecule, exposing 3 unpaired nucleotides on the rounded end which is opposite to the open end where the amino acid is attached. These three unpaired nucleotides on the tRNA make up an anticodon. Initiation: The smaller subunit has an affinity for mRNA, and the larger subunit has an affinity for tRNA. Special proteins called initiation factors guide the attachment of mRNA to the smaller subunit, and tRNA to the larger subunit. The two subunits then bind together to form an active ribosome (the initiation complex). The larger subunit contains two sites: the A site (amino acid or entry site), and the P site (also called D site--the polypeptide, peptide or donor site). The A site is for the reception of the incoming tRNA that carries an amino acid, and the P site is the holding place for the tRNA that carries an ever increasing length of amino acids. Only when the tRNA that carries an initiation amino acid, methionine, binds to the P site, can the protein synthesis begin. Elongation: The length of the amino acid sequence in a polypeptide is determined by the number of bases in mRNA. Three adjacent bases in mRNA form a triplet unit called a codon. Each codon codes for one amino acid (Table 9.3, p.271). Only two codons can attach to a ribosome at a time. Translation of the genetic code in the mRNA occurs from 5' to 3' direction. After the binding of tRNA that carries an initiation amino acid (methionine) at the P site, the second tRNA that also carries an amino acid enters the A site. The enzyme peptidyl transferase joins the carboxyl end of the first amino acid with the amino group of the second amino acid. The first tRNA then disconnects from the amino acid, and breaks off from the ribosome to pick up another amino acid. The second tRNA (in A site) which now has two amino acids at its end then vacates the A site and moves to the P site. The A site is now ready to accept the third tRNA that also carries an amino acid. In this manner, the polypeptide grows in length at a rate of about 15 amino acids (12-17 amino acids) per second until a specific termination codon is reached at the end of the mRNA. Many ribosomes can attach to a single mRNA to form a polyribosomal complex. Many polypeptides can be synthesized in tandem procession. Termination: Translation process stops when a termination codon (nonsense codon) is encountered. Special enzymes called release factors (also called termination factors) bind to the termination codon (UAA, UAG or UGA) and break the chemical bond that holds the polypeptide and the final tRNA, releasing it from the ribosome. The released polypeptide spontaneously folds to form a specific structure of the protein molecule. The mRNA is unstable. It is broken down by polynucleotide phosphorylase to its constitutive components: phosphate, ribose and nitrogenous base, which can be reused to synthesize other RNAs.

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A REPRESSIBLE OPERON 2. End-Product Corepression System (End-Product Repression or Gene Repression): (Fig. 9.22, p. 279) In this system, the repressor protein is inactive (not able to bind to the operator region) and the gene is turned on all the time to produce enzymes. The enzymes break down substrate into end product which serves as a corepressor substance. The end product binds to the repressor protein which then becomes activated. It is then able to bind to the operator region. The gene is turned off and no enzyme is synthesized. An example is the system of genes coding for enzymes necessary for arginine synthesis. The enzymes break down the substrate to form arginine (as a corepressor substance) which then binds to the repressor protein. The repressor is activated, it can then bind to the operator region. The binding of repressor to the operator region physically blocks the binding of RNA polymerase. Since the RNA polymerase cannot bind to the promoter region, no transcription can occur. The production of enzymes is then stopped. When the end product (arginine) is used up, the repressor protein changes its conformation and detaches from the operator region. Since the operator region is not blocked, the RNA polymerase can then attach to the promoter region again, and the gene is turned on. The oncogenes in cancer cells may synthesize a chemical which activates the gene that controls cell growth. The cell undergoes an uncontrolled cell division and turns into tumors and leukemias. ANTIBIOTICS THAT AFFECT TRANSCRIPTION AND TRANSLATION Certain antibiotics react with DNA, RNA and ribosomes, and thus alter genetic expression. Rifamycins bind to RNA polymerase and block the transcription. The drugs are selectively more active against bacterial RNA polymerase than the eucaryotic enzyme. Actinomycin D binds to DNA and halts mRNA elongation. Since its action is not selective, it is not used in treatment for bacterial infections except tumor treatment. Erythromycin and spectinomycin prevent translation by interfering with the binding of mRNA and ribosome. Aminoglycosides (such as streptomycin) inhibit peptide initiation and elongation. CHANGES IN THE GENETIC CODE: MUTATIONS AND INTERMICROBIAL EXCHANGE AND RECOMBINATION Mutation is a change in the base sequence of DNA. An organism that has undergone mutation is called a mutant. The parent organism with a normal genotype is called the wild type or prototroph. The mutant strain can be isolated with the replica plating technique (Fig. 9.23, p. 280). It is important to understand that the mutant strains are already in existence before the exposure of the bacteria to the selecting substance such as antibiotic. CAUSES OF MUTATIONS A mutation can be spontaneous or induced. A spontaneous mutation is a random change in the base sequence caused by errors in replication or natural background radiation. This type of mutation occurs one in every 105 to 1010 replications. An induced mutation is caused by purposeful exposure to mutagens. Acridine dyes such as proflavin or 5-aminoacridine resemble purines. They can insert (intercalate) themselves between adjacent base pairs, causing a frame-shift mutation, or the formation of nonsense codons and the elimination of termination signals.

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Analogs such as 2-aminopurine (2-AP) and 5-bromouracil (5-BU) are chemicals whose structures are similar to the nitrogenous bases. During DNA replication, they can be incorporated into DNA in place of the normal bases. Because of their unusual structures, they can cause errors in DNA replication. Base analogs are widely used in cancer treatment. They kill actively growing cancer cells because the DNA cannot be properly duplicated. Radiation energy can either kill a cell or cause a mutation depending on the duration of exposure and the dosage of irradiation. Ionizing radiation [X rays, a-rays (helium nuclei), y-rays and 0-rays (high speed electrons), neutrons, cosmic rays, and radioactive materials] induces simple base substitutions and large deletions. These rays also inactivate enzymes and RNA. Ultraviolet (UV) light has wavelengths ranging from 2,100 to 3,100 A (210-310 nm) with maximum bactericidal effect at 2,650 A. It causes dimerization (formation of dimers) between two adjacent pyrimidines (T-T, T-C or C-C) along the same polynucleotide. DNA with dimers cannot be properly transcribed or replicated as the dimers are skipped during the process. No mRNA can be properly transcribed from the DNA strand, and no DNA replication can take place. It often causes cell death. CATEGORIES OF MUTATIONS (Table 9.6, p. 283) Point mutation (base substitution or substitution mutation) is a change that involves only one base pair. This type of mutation may or may not cause a change in the amino acid sequence of a polypeptide. If the mutation results in no change in the amino acid sequence, it is known as a silent mutation. If the mutation results in a termination codon, it is known as nonsense mutation. This results in the formation of an incomplete or a defective protein which will cause a serious harmful effect on the organism. A point mutation may cause a displacement of a normal amino acid. This type of mutation is called missense mutation. Replacement of a normal amino acid rarely results in a complete inactivation of the protein. There are two types of point mutation: transition and transversion. Transition is a replacement of a purine (adenine or guanine) by another purine, or a pyrimidine (cytosine or thymine) by another pyrimidine. A-T G-C or C-G < = = = = = = = = = > T-A Transversion is a substitution of a purine (adenine or guanine) by a pyrimidine (cytosine or thymine) or vice versa in a DNA molecule. A-T C-G or C-G A-T A back-mutation refers to a type of mutation in which a gene undergoes a reverse mutation to its original base composition. Addition mutation: It is a type of mutation in which a base sequence has been added or duplicated. Deletion mutation: It is a type of mutation in which a base sequence has been eliminated from the DNA sequence. Addition and deletion may also be considered a point mutation if only one base pair is involved.

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67 Frame-shift mutation: Addition or deletion may cause a shift in the reading frame of DNA. It may give rise to a defective or non-functional protein. Inversion mutation: A base sequence is disconnected from the normal DNA. It turns around (reverses its polarity) and is reconnected to the same DNA. The sequence of bases along both strands of DNA are completely altered. Translocation: It is an exchange of segments of DNA between two heterologous chromosomes. Transposition: It is an insertion of a sequence of nucleotides (or block of genes) from one position to another in the same DNA. AAGGTATTCCGT ------------- > AAGTTCGTACGT REPAIR OF MUTATIONS Cells can produce repair enzymes to repair the DNA damage caused by mutagens. The repair enzymes remove (excise) the damaged base pairs or mispaired bases, and substitute them with the correct bases. If the repair requires visible light and a light-sensitive enzyme called DNA photolyase, this type of repair is known as photoactivation or light repair. If the damage is severe, no repair can be made and the cell will die. In humans, xeroderma pigmentosa is caused by non-functioning genes for photolyase. Severe skin cancers develop. THE AMES TEST (Fig. 9.26, p. 285) This test was developed in 1975 by Bruce N. Ames of the University of California at Berkeley. It is the standard test and is the most widely used. The test is based on the principle that mutagenicity and carcinogenicity are both caused by a change in DNA molecule. If a chemical is able to cause mutation, it is also a potential carcinogen. The test has been simplified to Ames spot test which is similar to antibiotic sensitivity test. The test organism is a mutant strain (auxotroph) of Salmonella typhimurium which has lost the ability to synthesize the amino acid histidine, a necessary component of proteins. In the absence of this amino acid the bacterium simply cannot grow because it lacks the necessary enzyme to synthesize this amino acid. The test is done by mixing the bacterium (about 1 billion cells) and rat-liver extract which is then plated on a histidine-deficient medium. The purpose of including rat-liver extract is to simulate the mammalian metabolic processes. Many chemicals require activating enzymes in the liver in order to become carcinogenic. A paper disc containing suspected carcinogen is placed on the medium previously inoculated. After incubation for two days, if bacterial colonies are found on the medium, it indicates that the mutant strain has already repaired its DNA and changed back to the normal type (prototroph) which does not require the addition of histidine in the medium. This reversion from the mutant strain to the normal strain is called reverse mutation. The number of colonies per mole of the tested chemical provides a quantitative estimation of the mutagenic potency of the chemical. An appropriate control has to be set up simultaneously to verify that the reverse mutation is caused by the chemical instead of spontaneous mutation. If a significant level of mutation occurs in the tested medium, it indicates that the chemical is potentially carcinogenic. About 90% of the chemicals tested and found to be mutagenic turned out to be also carcinogenic. POSITIVE AND NEGATIVE EFFECTS OF MUTATIONS Most spontaneous mutations are harmful to the organisms. A small number of mutations contribute to genetic variability which is essential for the evolution of organisms.

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INTERMICROBIAL DNA TRANSFER AND RECOMBINATION In eucaryotes, genetic recombination or hybridization through sexual reproduction is a source of genetic variation. In bacteria, DNA can be transferred from one cell to another. The resulting genetic recombination provides additional genes for drug resistance, poison production, new metabolic capability, increased virulence and adaptation to the environment. Genetic Recombination in Bacteria: Procaryotes have only a single chromosome. Genetic recombination occurs only when a piece of homologous chromosome is transferred from a donor cell to a recipient cell. There are three processes of genetic transfer in procaryotes: conjugation, transformation, and transduction. Conjugation is a direct genetic transfer from a donor cell into a recipient cell through a sex pilus (conjugative pilus). The number of sex pilus is 1 to 5 per cell depending on the growth conditions. The sex pilus is commonly found in Gramnegative bacteria but rarely in Gram-positive bacteria. This process requires the direct contact between two cells. The transfer of the genetic material is a one-way direction from the donor to the recipient. The bacterium that contained a genetic factor responsible for fertility called fertility factor (also called F factor, F particle or conjugon) is designated as F+, and the one without this genetic factor is designated as F . The F factor is a plasmid which is an extra piece of chromosome containing about 40 genes. The plasmid is not an essential structure for the survival of the bacterium. Only the bacterium that possesses F factor is able to produce sex pilus which is essential for conjugation (Fig. 9.27a, p. 288). When bacteria of two mating types come into contact, the sex pilus of F+ strain attaches to the cell membrane of F- strain. The pilus contracts, pulling two cells together. The plasmid in the F+ strain is immediately activated. A nick is made at the origin of the plasmid. One strand of the plasmid is transferred to the F- strain. As the linear strand of plasmid is being transferred, a new strand of the plasmid is synthesized for the displaced strand, restoring its duplex form. A new complementary strand of the plasmid is also synthesized in the recipient cell. Both strands are then closed by enzyme, forming a covalently linked circle of duplex DNA. The F- strains, once received a plasmid containing the F factor, is converted into F+ strain. Sometimes, a plasmid becomes incorporated into the host normal chromosome and becomes part of it. The plasrnid that can remain as autonomous chromosome or as part of the bacterial normal chromosome is called an episome. A bacterial cell with an integrated F factor in its normal chromosome is called a Hfr (high frequency recombination) cell . The probability of an F+ strain giving rise to Hfr strain is about 10-5 per cell (1 in 100,000 cells) per generation. It is so named high frequency recombination because the genetic recombination between Hfr x F- is 1,000 times greater than P x Fstrains. The rate of transfer is about 50,000 base pairs per minute. It takes about 100 minutes at 37°C for the entire strand of DNA to be transferred. Complete transfer of chromosome seldom happens because the cells are often interrupted and the pilus breaks. The F factor can excise from the chromosomal DNA and resumes autonomous replication. There are two types of excision of F factor. In type I excision, the F factor carries some bacterial genes as proximal or distal markers, and leaves some nonessential sequence of the F factor on the bacterial chromosome. This type of cell is designated as F' (F prime) cell. When this happens, rapid transfer of F factor can resume again. The F' factor is an autonomous unit and is able to exert its genetic influence. Because the F' factor carries bacterial genes, upon transfer to a

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F- cell, it can pair preferentially with the corresponding region of the recipient chromosome to form a Hfr strain. The transfer of chromosomal genes by the F' factor (a modified F factor) from a donor cell to a recipient cell is called sexduction or F-induction. The probability of this type of transfer giving rise to a Hfr strain is 10-1 per division cycle as compared to 10-5 for an F+ strain. A less common type of excision is the Type II excision which results in F factor picking up bacterial genes at both ends. The bacterial chromosome is left with no episome, and in addition, it also suffers a deletion for the markers gained by the episome. Deletion of markers from the chromosome may have a deleterious effect. The transfer of F factor may result in a recipient cell with certain traits in duplicate. When this cell divides again, the daughter cell again becomes a haploid cell with only one set of genes. Transformation is a process of genetic transfer in which the DNA released from a donor cell into the environment is absorbed and incorporated into the chromosome of a recipient cell. The recipient cell becomes a mutant as it acquires new genetic traits from the donor cell. Transformation is the first type of genetic transfer in bacteria observed in 1928 by Frederick (Fred) Griffith, an English pathologist and medical officer in the British Ministry of Health. He reported that when the mice were injected with a mixture of the dead cells of Type III virulent pneumococcus (Streptococcus pneumoniae) and live Type II avirulent pneumococcus, the mice would die of pneumonia (Fig. 9.28, p. 289). Both the live Type H and the killed Type III pneumococci did not kill the mice when injected separately. The Type II pneumococcus produces no capsule, and it gives rise to pinpoint colonies which appear rough on culture medium. Hence, it is designated as R-type. The Type III pneumococcus produces capsule and gives rise to large and smooth colonies. It is designated as S-type. Capsule is responsible for pathogenicity as it prevents phagocytosis by white blood cells. The ability to produce capsule is due to the presence of a gene responsible for its production. He proposed that a transforming principle (factor) released from the killed pneumococcus was responsible for transforming the R-type into the S-type. The chemical nature of the transforming principle was identified as DNA in 1944 by Oswald T. Avery, Colin Mac MacLeod and Maclyn McCarty at the Rockefeller Institute fcr Medical Research (now Rockefeller University) in New York city. A cell which is able to absorb donor chromosome is called a competent cell. Absorption of DNA fragments occurs only in the late logarithm phase before a population enters the stationary phase of growth. The competence phase may last for several minutes to several hours depending on the growth conditions and species. In a competent cell, a competent factor is produced. It is a protein of 10,000 daltons which can be transferred to a non-competent cell. The competent factor causes a limited release of autolysin into the periplasmic space between cell wall and cell membrane of a recipient cell. The autolysin causes the cel l wall to weaken. Once the cell wall is weakened, the transforming DNA can attach to the exposed receptors on the cell membrane, and is transported into the cell by mesosome. After entry, the integration of the transforming DNA (1,500-8,000 bases) takes place fairly rapidly. Transformation is reported in the closely related strains of the following organisms: Escherichia coli, Shigella paradysenteriae, Bacillus subtilis, Hemophilia influenzae, Neisseria meningitidis, Staphylococcus, Streptococcus, Acinetobacter, Rhizobium, yeasts and mammalian cells. Transfection is the uptake of viral DNA in the host cell. The absorbed DNA can be replicated into many transfecting DNA called transgenomes which are unstable in most cells. The viral DNA can be incorporated into the chromosomes of a small percentage of cells.

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Transduction: The Case of the Piggyback DNA Transduction is the transfer of genetic material from a donor cell to a recipient cell by a virus (Fig. 9.29, p. 290). It was accidentally discovered in 1952 by Norton D. Zinker and Joshua Lederberg then at the University of Wisconsin. There are two types of transduction: general (generalized) and restricted (specialized). In general transduction, all the genes in a host chromosome have an equal chance to be transferred to another cell by a virus. This occurs when a virus invades a cell and carries out a lytic cycle. The host chromosome is broken up into small pieces which might be included as part or as the sole chromosome free from viral chromosome within the viral protein coat. When the virus invades another cell, it will donate the donor chromosome to the recipient cell. During bacteria reproduction, crossing over occurs between the donor and the host chromosomes, resulting in genetic recombination. Another type of transduction is called restricted or specialized transduction. In this type of transduction, a bacteriophage chromosome, after injection into a host cell, may remain as an episome or becomes incorporated into the host chromosome at a particular region. This process of genetic incorporation is called lysogeny. The piece of bacteriophage chromosome in the bacterium (lysogenic bacterium) is called a prophage or lysogen. The prophage remains latent in the host cell because of the presence of a repressor protein synthesized by the prophage. It blocks the expression of the viral genes. The presence of prophage confers immunity to superinfection of the host cell because of the repressor protein. The prophage can be replicated and passed on to the next generation only when the host cell divides. At time, the prophage may become active again when it is exposed to ultraviolet light, increased temperature, mitomycin C or other mutagenic agents which inactivate the repressor. Errors may occur during the detachment of the prophage from the host chromosome. A prophage may lose part of its chromosome and include part of the bacterial chromosome instead. Only the bacterial genes adjacent to the prophage can be included. When this virus invades other bacteria, it introduces the donor chromosome into the host cell. The virus becomes lysogenized in the host cell, imparting new hereditary traits to the cell. The virus cannot multiply in the host cell because it has part of its genes missing. It can multiply only when a new virus which serves as a helper virus invades the cell and furnishes its missing genes. Transposons: "This Gene is Jumping" Transposons are also called jumping genes because they can jump from one DNA to another, resulting in mutation of the cell. They were first discovered in 1951 by Barbara McClintock whose significant discovery was ignored by scientific community for many years. She was awarded the Nobel Prize in 1984. She was one of the three scientists who were ever awarded the unshared Nobel Prize. Transposons can be found in plasmid or chromosomal DNA. They contain genes for the production of transposase which cleaves DNA insertion sequence, and repressor that regulates its activity. They can cause rearrangement or deletion of genes from the chromosome. They also carry genes that code for antibiotic resistance, degradation of organic compounds, enterotoxin and changes in colony morphology, pigmentation, pili and antigenic characteristics. Since they can be transferred even between unrelated organisms, they create genetic diversity, essential for the evolution and survival or organisms. HIV behaves like a transposon as it randomly insert in the genome that leads to severe cell dysfunction.

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Chapter 10

GENETIC ENGINEERING: A Revolution in Molecular Biology BASIC ELEMENTS AND APPLICATIONS OF GENETIC ENGINEERING Recombinant DNA technology or genetic engineering is the method of introducing the genes from one organism into another such as from animal or plant into bacteria in the laboratory. Various biological products have been produced by this technique. Human insulin gene has been introduced into Escherichia coli to synthesize the human insulin. Many diabetic patients have to use human insulin as they react against the foreign bovine insulin. The topics are divided into six major sections: 1. DNA tools, (2) Recombinant DNA technology, (3) recombinant products, (4) transgenic organisms, (5) genetic treatments, and (6) genetic analysis. (Fig. 10.1, p. 300) TOOLS AND TECHNIQUES OF GENETIC ENGINEERING DNA: THE MARVELOUS MOLECULAR TOY DNA can be denatured by heat. When it is exposed to 90° to 95°C, the hydrogen bonds that hold the two strands break, forming two strands. These two strands will anneal when the temperature is lowered to about 30°C (Fig. 10.2, p. 301). This property allows scientists to amplify DNA. Enzymes for Discing, Splicing, and Reversing Nucleic Acids: A restriction endonuclease is an enzyme that cleaves DNA at a specific sequence (a sequence with 4 to 10 base pairs). The enzymes are produced by nearly all microorganisms. Their function is to identify and destroy foreign DNA that might have entered the cells. They do not cut their own DNA because the specific DNA sequence to be cleaved by the enzyme is modified by methylation (methyl groups, -CH3, are added to the nucleotides within the sequence). Some restriction endonucleases cut the DNA into fragments with blunt ends. Some enzymes cut the DNA with sticky ends which have palindromic sequences. The double stranded DNA fragments with protruding single strands at both ends have complementary bases. The ends will automatically rejoin if the enzyme is removed. If a foreign DNA is cut by the same enzyme, DNA fragments with identical sticky ends will be formed. When the plasmids and the DNA fragments are mixed, and DNA ligase and ATP are also added, they will join together to form a recombinant DNA (rDNA) (Fig. 10.3, p. 301). Fragments of DNA produced by restriction endonucleases are called restriction fragment length polymorphisms (RFLPs) which serve as a genetic marker. Since each individual has different genome, they are useful in preparation of gene maps and DNA profile (DNA fingerprinting) (Fig. 10.18, p. 320 and Microfile 10.4, p. 321). Ligase is a sealing enzyme which joins the phosphate-sugar bonds cut by endonucleases during gene splicing. Reverse transcriptase is found in HIV and other retroviruses. It uses RNA (transfer, ribosomal or messenger) as a template to synthesize a copy of complementary DNA (cDNA). It allows scientists to synthesize eucaryotic gene free of introns.

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Visualizing DNA with the Naked Eye: DNA fragments can be separated by gel electrophoresis (Fig. 10.4, p. 301). The separation is base d on the electrical charges and molecular sizes. Samples are placed in wells on a gel and subjected to an electric current. The cations (+) will migrate to cathode (-), and anion (-) will migrate to anode (+). In this manner, the DNA fragments can be separated. The position of DNA fragments can be visualized by stain or a labeled probe. The patterns of DNA fragments (DNA fingerprint) appear as bands which can be compared to see the degree of genetic similarities. Methods Used to Size, Synthesize and Sequence DNA: E. coli has about 4.7 million base pairs (bp) and humans have about 3.5 billion base pairs in 46 chromosomes. It is difficult to analyze large segments of DNA or RNA. Oligonucleotides are short chains of nucleotides. They vary from 2 to 200 base pairs. The most common ones are about 20 to 30 base pairs. The precise sequence of a DNA can be read by machines called sequencers. They can read the DNA sequence at a rate of thousands nucleotides per day. Nucleic Acid Hybridization and Probes: The presence of a specific DNA or RNA sequence can be detected with a labeled probe which can be either DNA or RNA. Their base sequences have to be complementary in order for them to be hybridized (paired or connected). In a technique called the Southern blot, the DNA fragments are first separated by electrophoresis and then denatured and immobilized on a nitrocellulose paper. A labeled DNA probe is then incubated with the sample. If their base sequences are complementary, they will join together. The hybridization pattern will show up as one or more bands (Fig. 10.4, p. 301). Probes can be used to identify an unknown bacterium or virus. In the dot blot method, an unknown sample is isolated, denatured, placed on an absorbent filter, and combined with a specific labeled probe (Fig. 10.5, p. 303). Identification can be made when the blot is developed and observed for hybridization. Diagnostic kits are available for identifying a number of diseases caused by bacteria and viruses. In another method called in situ hybridization, probes are applied to intact cells to visualize the presence and location of specific nucleic acid sequences. It can also be used to identify bacteria without having to culture them. Polymerase Chain Reaction: A Molecular Xerox Machine for DNA The polymerase chain reaction (PCR) is a technique used to amplify the DNA in vitro. It was first introduced by scientists at Cetus in 1985. It requires primers (synthetic oligonucleotides with 15 to 30 bases), and DNA polymerases to synthesize new DNA. The Taq DNA polymerase is isolated from thermophilic bacterium Thermus aquaticus and the Vent polymerase is isolated from Thermococcus litoralis. These thermostable enzymes allow the primers to be annealed and extended at much higher temperature. The reaction is operated on three basic steps: denaturation, priming and extension. The target DNA is denatured by increasing temperature from 94°C to 98°C. Under these high temperatures, the hydrogen bonds that connect these two strands break apart. A double-stranded DNA then forms two single-stranded DNA. Each strand is called an amplicon. The primer anneals to the 3' hydroxyl end of each strand at 37°C to 65°C. The annealed primers are then extended on the template strand by a DNA polymerase from 5' to 3' direction at 72°C. A thermal recycler automatically initiates the cyclic temperature changes. In this manner, a DNA molecule forms two DNAs. The three steps: denaturation, primer binding and DNA synthesis, represents one PCR cycle. Repeated cycles of

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denaturation, primer annealing, and primer extension result in the exponential formation of DNA molecules (Fig. 10.6, p. 304). A PCR machine can perform 20 cycles on nearly 100 samples in an hour or less. The amplified DNA can be directly used for electrophoresis, probe identification and sequencing. PCR can be used to analyze RNA by first converting RNA into cDNA with reverse transcriptase. The cDNA can then be amplified and analyzed. The technique plays a vital role in gene mapping, the study of genetic defects and cancer, forensics, taxonomy and evolutionary studies. The major problem with PCR is the possible contamination of the target DNA. This can be solved by using very specific primers and special enzymes to digest the contaminating DNA before it is amplified. METHODS IN RECOMBINANT DNA TECHNOLOGY: HOW TO IMITATE NATURE The following are the basic recombinant DNA procedures: (Fig. 10.7, p. 305) 1. DNA of interest is isolated from a donor organism, or directly synthesized in the laboratory. 2. Plasmid DNA is isolated from a bacterium. It is used as a vector to carry the desired gene. 3. Both the DNA and the plasmid are treated with the same restriction endonuclease. The enzyme cuts the DNA and the plasmid in such a manner that both have complementary single-stranded ends (sticky ends). 4. When both the DNA fragments and the plasmid are mixed, they form recombinant plasmids (recombinant DNA, rDNA). They can join because the bases on the sticky ends are complementary. 5. The recombinant plasmid is added to a suspension of bacteria which take up the plasmid by transformation. 6. The bacteria are then isolated in pure cultures, and the colonies that synthesize the product are identified. 7. The genetically engineered bacteria are cloned in large quantities. The product can be recovered from the cultures and purified. TECHNICAL ASPECTS OF RECOMBINANT DNA AND GENE CLONING A desired gene can be obtained from: 1. The DNA is removed from the cells that have the desired characteristic. The DNA is cleaved by endonucleases. Each fragment is inserted into a vector and cloned. The cloned fragments are Southern blotted to identify the desired sequences. But this process is tedious. If human genome were separated into 20 kb fragments, there would be at least 150,000 clones to detect. 2. A gene can be synthesized from mRNA using reverse transcriptase. 3. DNA can be synthesized by a machine. But the size of DNA is limited. Once a gene has been isolated and cloned, it can be maintained for ever. A collection of these cloned genes from an organism constitutes a genomic library of that organism. Characteristics of Cloning Vectors: A good cloning vector should be able to carry a significant piece of the donor DNA, and it must be readily accepted by the cloning host. The cloning vector can be a plasmid, a bacteriophage, a cosmid or the yeast artificial chromosome (YACs). Plasmids are excellent vectors because they are small, well characterized, easy to manipulate and can be transferred into appropriate host cells through transformation. The plasmids that carry genetic markers for antibiotic resistance have the advantage that any cells that pick up the recombinant DNA will be resistant to antibiotics. This allows elimination of cells that do not pick up the recombinant DNA. However, the plasmid can accept only a small amounts of foreign DNA. Bacteriophages can inject DNA into bacteria through transduction. They too can accept only a small amounts of foreign DNA. A modified

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phage vector called the Charon phage has large sections of its genome deleted, so it can carry a fairly large segment of foreign DNA. Another vector is called cosmid which is a hybridization between a plasmid and a phage. It is capable of carrying relatively large genomic sequences. A hybrid E. coli-yeast vector can be inserted into the bacterium and the yeast. The yeast artificial chromosomes (Y A Cs) are plasmids from the common brewer's yeast Saccharomyces cerevisiae. They can carry 10 times the amount of DNA compared to the common plasmids. They are able to replicate more complex genome outside their natural host. Characteristics of Cloning Hosts: (Table 10.1, p. 307) The desirable characteristics of a cloning host are rapid growth rate, able to grow in large quantities using ordinary methods, nonpathogenic, genome is well mapped, capable of accepting plasmid or bacteriophage vector, able to maintain foreign genes through generations, and able to secrete a high yield of gene product. The common cloning hosts are E.coli, Bacillus subtilis, and Saccharomyces cerevisiae. CONSTRUCTION OF A CHIMERA, INSERTION INTO A CLONING HOST, AND GENETIC EXPRESSION The following steps show the recombinant DNA technique to produce alpha-2-interferon (Roferon-A). This interferon is used to treat cancers such as hairy-cell leukemia and Kaposi's sarcoma. The gene is prepared from mRNA transcript free of introns from human red blood cells. It codes for a polypeptide of 166 amino acids. The gene is attached with terminal nucleotides (sticky ends) complementary to the sticky ends of an E. coli plasmid which has been cleaved with an endonuclease (HindIIl). When they are mixed and with the help of a DNA ligase, they splice to form a chimera (a recombinant plasmid). The chimera is introduced into E. coli that lacks plasmids. The recombinant plasmid enters the cell by transformation. Most bacteria do not readily pick up plasmid in this manner. A method known as the calcium chloride (CaCl2 ) transformation procedure has proved effective. The recipient cells are treated with CaC12 under cold temperature (0° to 4°C for E. coil), and then warmed (to 42°C for E. coli) to heat-shock the cells. The treatment alters the permeability of the plasma membrane which then permits the entrance of the plasmid. Another method is called electroporation which uses an electric shock treatment. The short electric pulse causes the formation of pores which allow the diffusion of the plasmid. The plasmid that contains gene responsible for ampicillin resistance is selected to be used as a vector. When the bacteria are plated on the medium that contains ampicillin, only those cells that have picked up the plasmid can grow (Fig. 10.10, p. 308). Those do not pick up the plasmid will die. The colonies are cloned. Large quantities of interferon can be produced by this cloned gene. The gene product can be extracted and purified (Fig. 10.9, p. 307).

BIOCHEMICAL PRODUCTS OF RECOMBINANT DNA TECHNOLOGY Recombinant human insulin (Humulin) is used to treat diabetics, and recombinant HGH (Protropin) is used to treat dwarfism in children. Vaccines and other biological products have been successfully produced by genetic engineering techniques (Table 10.2, p. 309). RECOMBINANT ORGANISMS: HOW TO IMPROVE ON NATURE Transfection is the process of introducing foreign genes into organisms. The recombinant organisms are called transgenic. The transgenic organisms can be patented.

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RECOMBINANT MICROBES: MODIFIED BACTERIA AND VIRUSES One of the first practical application of rDNA in agriculture was the use of genetically engineered Pseudomonas syringae whose gene responsible for ice nucleation had been eliminated. Frost damage in agriculture is due to the ice crystal formation around the nucleation protein produced by the bacterium. A gene from Bacillus thuringiensis responsible for toxin production has been introduced into Pseudomonas fluorescens. This genetic engineered bacterium is introduced into plant roots to destroy insects. The release of any genetic engineered organisms must be approved and monitored by the Environmental Protection Agency (EPA). So far, there is no problem. A genetically engineered Rhizobium had been approved by the EPA to be released to increase the ability of plants to fix nitrogen. However, due to concerns about the possible spread of the genes to other plants and the creation of superweeds, the release was blocked. The genes responsible for luciferase have been isolated from fireflies and inserted into a bacteriophage. The enzyme causes light emission. The virus is allowed to infect Mycobacterium tuberculosis which is then used in drug tests. If the bacterium is not killed by the drug, it will light up (Fig. 10.12, p. 310), indicating that it is resistant to the drug. If it dies no light is emitted, indicating that the drug is effective. The advantage of this type of test is that it takes only a few days instead of several weeks normally required. Genetic engineered viruses have been developed for use as vaccines, for gene therapy, insect pest control and degradation of environmental pollutants such as oil spills, pesticides, and toxic substances. TRANSGENIC PLANTS: IMPROVING CROPS AND FOODS Agrobacterium tumefaciens causes crown gall disease in plants. It contains a plasmid called Ti (tumor inducing) plasmid. When it infects plants, the plasmid inserts into the DNA of the infected cells and transforms them. This plasmid has been used as a vector to introduce foreign genes into plant genomes. For those plants that cannot be transformed with this bacterium, gene guns can be used to shoot minute gold pellets coated with the desired genes directly into plant embryos. This technique is used to create pesticide resistant and pathogen resistant plants (Fig. 10.14c, p. 312). Another technique for engineering plants is to insert an antisense RNA that can turn off the expression of a target gene (Fig. 10.17, p. 316). The oligonucleotide strands of RNA or DNA enter the nucleus and bind to mRNA that codes for enzymes responsible for tomato ripening. Such bound RNA cannot leave the nucleus, and no translation can occur. The tomato does not ripen too rapidly and can be left on the vine to develop its natural flavor. The antisense technology is also applied to develop a rapeseed (canola) oil plant with more saturated fat, making it suitable to make margarine without artificial hydrogenation. Tobacco plants have been engineered to synthesize human antibodies which can be used in diagnosis and treatment of diseases. TRANSGENIC ANIMALS: ENGINEERING EMBRYOS Transgenic animals can be created by introducing foreign genes into their genomes. The animals are used as models to study human genetic disease mechanisms, and to test genetic therapies prior to their use in humans. Fertilized egg or early embryo is transfected with foreign genes by pulsing it with high voltage to enable the gene to enter the cell membrane. Very few embryos can survive with this treatment. Once a transgenic animal is created, it will pass its genes on to its progeny. Mouse embryos injected with human genes for growth hormone develop into super mice twice the size of normal mice (Fig. 10.15a, p. 305). The "knock out mouse" can be created by transfecting a mouse embryo with a defective gene and cross breeding the progeny through several generations.

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The mice with defective genes will express the disease. Mouse models have been developed for cystic fibrosis, hardening of arteries, spondyloarthropathy (an inflammatory disease with symptoms of arthritis and psoriasis), Gaucher's disease (a lysosomal storage disease), and Alzheimer's disease. In animal husbandry, pig embryos have been implanted with the bovine growth hormone gene. The transgenic pigs produce less fat (Fig. 10.15c, p. 313). Unfortunately, it also contributes to severe health problems (arthritis and heart and kidney disease). The pharmaceutical industry has engineered a strain of transgenic pig to produce clotting factors and human hemoglobin; a strain of sheep to produce alpha antitrypsin (for emphysema treatment) and clotting factors; and a strain of goats to produce cystic fibrosis membrane protein in their milk (Fig. 10.15c, p. 313). Engineered animals such as pigs will be used as living factories to grow tissues and organs (heart and kidney) for human organ transplant. GENETIC TREATMENTS: INTRODUCING DNA INTO THE BODY Gene therapy is a technique used to replace a defective gene with a normal gene in people with genetic diseases. There are two strategies: (1) In ex vivo therapy, the normal gene is cloned in vectors such as retroviruses (mouse leukemia virus) or adenoviruses that can cause infection but are harmless to humans. Tissues are removed from the patients and are incubated with the viruses to transfect them. The infected cells are then reintroduced into the patient by transfusion (Fig. 10.16, p. 314). (2) In vivo therapy, the virus vector is directly introduced into the tissues or organs. No excision of the tissues or incubation of the tissues with the virus is required. Gene therapy has encountered the following difficulties: (1) The systems used to deliver genes are not completely successful. (2) The rate of transfection by virus is very low (about 10%). (3) A safety concern is raised because the virus could insert the DNA at the wrong site and activates an oncogene or other dormant viruses. Alternate delivery systems are the use of gene guns, liposomes, and dendrimers. Liposomes are tiny sphere with lipid bilayer and aqueous center that can carry the genes across the membranes. Dendrimers are very large, branched hydrocarbon molecules that can transfer genes into cells. The DNA picked up by some cells may remain free in the nucleus of the transfected cells. The cells can translate the gene for a limited time. But the gene may not be duplicated and passed it on during cell division. Scientists are working on transfecting the stem cells taken from the umbilical blood of newborns. The cells may be able to pass the gene on and correct the genetic defects. The first gene therapy was performed by researchers at the National Institutes of Health on a 4-year old girl with a severe immunodeficiency disease. The disease is caused by the lack of the enzyme adenosine deaminase (ADA). She was transfused with her own blood cells which had been transfected with the normal ADA gene. Later, another child was similarly treated. Both patients showed dramatic improvement. But they still have the disease. Adenoviruses engineered to carry normal cystic fibrosis gene were directly delivered into the nose and lungs of the patients. Early results indicate that the epithelial cells of the organs appear to accept the gene and function normally. Table 10.3 (p. 315) summarizes some human genetic defects targeted for gene therapy. Gene line therapy using genetic engineered egg, sperm or early embryo may offer promise in animals but controversial in humans.

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ANTISENSE AND TRIPLEX DNA TECHNOLOGY: GENETIC MEDICINES The genetic drugs like antisense (DNA or RNA) and triplex DNA are oligonucleotides which can be delivered into the nucleus to block transcription or translation of a specific gene. This type of treatment bypasses the problem gene and its product. Antisense DNA: Targeting Messenger RNA Antisense refers to a nucleotide strand which has a base sequence complementary to that of the sense strand. For example, mRNA is a translable sense strand. If its base sequence is AUUCCG then its antisense strand is TAAGGC (or UAAGGC). Most antisense agents are DNA because the technology of making large-scale RNA is less feasible. Antisense molecules are modified by substituting one oxygen atom of phosphate with a sulfur atom. This modification makes the agents soluble and facilitates their uptake by cells. The antisense will bind to specific sites on any mRNAs and block their translation. Experiments in animals indicate the antisense drugs can block translation of an undesirable protein. The drawbacks of the drugs are the high cost (need to administer for life), inability to deliver a significant dose into cells, and the possibility of causing cancer or other diseases. This technology will not work on sickle-cell anemia and cystic fibrosis which are caused by a lack of a functioning gene. It may work on cancers, autoimmune diseases (multiple sclerosis), hepatitis B, AIDS and Alzheimer's disease (Table 10.3, p. 315). It has been used in tomato to block the gene that controls fruit softening. Triplex DNA: Three's a Crowd A triplex DNA is a DNA that has a third strand inserted into the major groove of the molecule. The inserted strand forms hydrogen bonds with the purines of one of the adjacent strands (Fig. 10.11b, 'p. 316). So far, only oligonucleotides of this type of DNA have been synthesized. They interfere with the binding of transcription factors and RNA polymerase, and thus prevent gene transcription. This molecule has been shown to block the genes coding for oncogenes, viruses, and the receptor for interleukin-2. This therapy may have the same problems with the antisense therapy. GENETIC MAPS, FINGERPRINTS, AND FAMILY TREES GENOME MAPPING AND SCREENING--AN ATLAS OF THE GENOME Gene mapping is the process of marking the relative order of genes on the chromosomes. Sequence map:; show the exact order of bases along the DNA. Up to now, only certain viral genomes and the bacterium Haemophilia influenzae have been completely mapped and sequenced. The gene maps for Escherichia coli, Saccharomyces cerevisiae and Caenorhabditis elegans (a roundworm) are near completion. The federal government started the human genome project (mapping of human genome) in 1986. The project is estimated to take 20 years to complete at a cost of 3 billion dollars. Human cell has a total of three billion base pairs and an estimated 100,000 genes which account for only 3% of the total amount of DNA. The other 97% of DNA was originally thought to be junk DNA. This type of DNA may function as chromosome stabilizers, gene regulators, attachment sites for mitotic fibers, and in ribosome assembly. If all the bases of the 46 chromosomes are printed out, a book with 1 million pages will be produced. This book is like a human gene dictionary or encyclopedia of humans. High speed computers are used to sort out the meanings of

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all these bases. So far, only 7,000 genes have been correlated with a specific location of a given chromosome, and 50 out of 5,000 inherited diseases have been located. The human gene map will reveal the risks for certain diseases. It will guide decisions on correcting or alleviating them through appropriate therapy. DNA FINGERPRINTING: A UNIQUE PICTURE OF A GENOME DNA fingerprinting (also called DNA typing or profiling) is a method of identifying an unknown DNA source with a known DNA. It is used in forensic investigation, identification of the risk of hereditary diseases, clarifying human paternity and maternity, the pedigree of animals, and the genetic diversity of animals, and in comparative and evolutionary studies. Fig. 10.18 (p. 320) shows the pedigree analysis of Alzheimer's disease. The site for this disease is on chromosome #14. Analysis of the DNA fragments cleaved by restriction endonuclease shows that A and C have the disease. They all share band 1. E and F also have band 1. So, they are at high risk for developing the disease.

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Chapter 11 PHYSICAL AND CHEMICAL CONTROL OF MICROBES CONTROLLING MICROORGANISMS Microorganisms can be destroyed by physical agents such as heat or radiation or chemical agents such as disinfectants, antiseptics or drugs. Figure 11.1 (p. 329) summarizes the major applications and aims in microbial control. RELATIVE RESISTANCE OF MICROBIAL FORMS The relative resistance to physical and chemical agents are as follows: Highest resistance: Bacterial endospores. Moderate resistance: Protozoan cysts, fungal sexual spore (zygospores) and some viruses (hepatitis B virus, poliovirus), Mycobacterium tuberculosis, Staphylococcus aureus and Pseudomonas species. Least Resistance: Most bacterial vegetative cells, fungal spores and hyphae, enveloped viruses, yeasts and trophozoites. Table 11.1 (p. 330) shows the requirements to destroy various groups of microorganisms. TERMINOLOGY AND METHODS OF MICROBIAL CONTROL Sterilization: A process of killing all forms of life. Any material that has been subjected to this process is said to be sterile. Sterilant: A chemical used for sterilization, particularly the heat-sensitive medical supplies. Microbicidal agents: Substances that kill microorganisms. Bactericidal agents (bactericides): Substances that kill bacteria. Fungicidal agents (fungicide): Substances that kill fungi. Antimicrobial agents: Substances that kill or inhibit the growth of microorganisms. Antibacterial agents: Substances that kill or inhibit the growth of bacteria. Virucidal agents (virucide): Substances that kill viruses. Antiviral agents: Substances that kill or inhibit the growth of viruses. Antifungal agents: Substances that kill or inhibit the growth of fungi. Antiprotozoan agents: Substances that kill or inhibit the growth of protozoa. Sporicides: Substances that kill spores. Bacteriostasis: A condition in which the bacterial growth is inhibited. Bacteriostatic agents: Substances that inhibit the growth of bacteria. Microbiostatic agents: Substances that inhibit the growth of microorganisms. Fungistatic agents: Substances that inhibit the growth of fungi. Germicides (microbicides): Chemicals that kill the vegetative forms of microorganisms. Disinfection: A process of destroying infectious agents. Disinfectant: A chemical agent used to destroy infectious agents associated with inanimate objects. Sepsis: Poisoning caused by the products of a putrefactive (decomposition) process. Asepsis: Absence of pathogens or their poisonous products. Aseptic: Free from living organisms. Antisepsis: Prevention of infectious diseases by inhibiting or killing of pathogens. Antiseptic: An agent that prevents the growth of microbes associated with the living body. Degermation: A procedure of reducing the numbers of microbes on the human skin by scrubbing, application of alcohol or cleansing with germicidal soap and water.

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Sanitization: A process of reducing the microbial number on inanimate objects to a safe level specified by public health standards. Sanitizes. A chemical agent that reduces the microbial number to a safe level. It is applied to inanimate objects, such as glasses, dishes, and utensils in restaurants, and food-processing plants. Preservative: A chemical that prevents the deterioration of biological products. Deodorant: A chemical that destroys or masks offensive odors. Astringent: A drug that acts locally on the cell surface by precipitating proteins. The cell remains viable. Fumigation: The destruction of insects or small animals by fumes or gasses. Fomite: A fomite is an inanimate object that can transmit a disease indirectly from person to person. The pathogen cannot multiply on the fomite. Vehicle: A vehicle is a substance that transmits a disease indirectly from person to person. The pathogen can multiply in a vehicle. WHAT IS MICROBIAL DEATH? Microbial death is the permanent loss of reproductive capability even under optimum growth conditions. Factors That Affect Death Rate of microorganismsms: (Fig. 11.2, p. 332) 1. Time of exposure to the microbicidal agent: Longer exposure kills more cells. 2. The number of microorganisms. It takes a longer time to kill a larger population. 3. The nature of the microorganisms (bacteria, fungi, spores or viruses or the mixture) in the population. Gram-positive bacteria are more resistant to heat than Gram-negative bacteria. Some chemicals are more effective against Gram-positive than Gram-negative bacteria. 4. Physiological state of microorganisms (young, old or spores). Young active cells are more susceptible than older cells or spores. 5. Temperature and pH of the environment. Higher temperature, and low or high pH kill microorganisms more rapidly. It takes a shorter time to kill microorganisms in a medium at a lower pH or a higher pH than a medium at a neutral pH. 6. Concentration (intensity) of the microbicidal agent: With a lower concentration, it may take a longer time to kill a microbial population. UV radiation is most effective at 260 nm. 7. Nature of the material containing the microorganisms: The presence of organic matter, physical state of medium (liquid, viscous or solid), inhibitors, saliva, blood and feces can inhibit the actions of disinfectants or heat. 8. Characteristics of the microorganisms which are present: Microorganisms exhibit differential susceptibility to antimicrobial agents.

HOW ANTIMICROBIAL AGENTS WORK: THEIR MODES OF ACTION The modes (mechanisms) of action refer to how microorganisms are removed or destroyed. There are five general categories: 1. Inhibition of Cell Wall Synthesis: Examples: penicillin, lysozyme, lysostaphin, detergents and alcohols. 2. Damage the Cell Membrane: Examples: surfactants, nystatin, amphotericin B. 3. Inhibition of Protein Synthesis:

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Examples: Chloramphenicol, streptomycin, tetracycline, erythromycin. 4. Inhibition of Nucleic Acid Synthesis: Examples: X ray, formaldehyde, ethylene oxide, actinomycin D, griseofulvin, and rifampin. 5. Alteration of Proteins: Examples: moist heat, alcohols, acids, alkalines, phenolics, and metallic ions. Practical Concerns in Microbial Control: 1. Does the microbial control require sterilization or disinfection? 2. Is the item to be reused or discarded? 3. Can the item withstand heat, pressure, radiation or chemicals? 4. Is the control method suitable? 5. Will the agent penetrate the material? 6. Is it cost effective? METHODS OF PHYSICAL CONTROL I. HIGH TEMPERATURES A. Moist Heat: This process destroys microorganisms by denaturation and coagulation of proteins. It is more effective than dry heat in destroying microorganismis. It takes 2 to 15 minutes to kill the endospores of Bacillus anthracis by moist heat at 100°C. Dry heat takes 180 minutes at 140°C. Moist heat kills vegetative cells in 5 to 10 minutes at 60° to 70°C. Moist heat kills the vegetative cells of fungi in 5 to 10 minutes at 50° to 60°C, and the fungal spores in the same time interval at 70° to 80°C. 1. Steam under pressure: The apparatus used is an autoclave (Fig. 11.5, p. 338). It is normally operated at a pressure of 15 lbs/in2 at 1210C for 15 minutes. 2. Boiling: It is effective against vegetative cells but not so much against spores. The spores of many saprophytes can survive for many hours of boiling. Hepatitis viruses are destroyed after 30 minutes of boiling. 3. Subboiling Temperatures: Pasteurization: It is a method of heating milk and dairy products to destroy harmful germs that cause tuberculosis, brucellosis (undulant fever), Q fever, streptococcal infections, staphylococcal food poisoning, salmonellosis, shigellosis, and diphtheria. Mycobacterium tuberculosis is destroyed at 60°C for 15 minutes. Low Temperature Holding (LTH) Method (Vat Pasteurization, Holding or Batch Method): 63°C (145°F) for 30 minutes. High Temperature Short-Time (HTST) Method (Flash Method, Continuous-Flow Method): 72°C (161°F) for 15 seconds. Ultra-High-Temperature (UHT) Sterilization: 134°C (some books say 141°C) for 1 to 2 seconds. It is used to sterilize milk, fruit juices, and drinks. No refrigeration is needed after sterilization. 4. Fractional sterilization (Tyndallization): The materials which cannot withstand a temperature over 100°C are subjected to free flowing steam for 30 minutes on three successive days with incubation periods in between. Resistant spores germinate during the incubation periods. The vegetative cells will be killed on subsequent exposure to heat. Gelatin, milk and sugar broth are sterilized with this method. 5. Inspissation (Thickening through evaporation): It is used for high protein-containing media that cannot withstand high temperatures of the autoclave. The media are placed in the Arnold sterilizer or inspissator at 75° to 80°C for 2 hours on three successive days. LowensteinJensen medium used for cultivation of mycobacteria, and Fletcher's medium for

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cultivation of Leptospira are sterilized with this method. Measurement of Microbial Susceptibility to High Temperatures: To determine the following values, the nature of the medium, the pH, and the initial concentration of microorganisms must be rigidly controlled. 1. Thermal death time (TDT): The shortest time within which a suspension of bacteria is killed at a given temperature. 2. Thermal death point (TDP): The lowest temperature at which a suspension of bacteria is killed in 10 minutes. B. Dry Heat: This process destroys microorganisms by dehydration. It is not as effective as the moist heat. It takes longer to kill the organisms. Dry heat is used for materials which cannot be sterilized with moist heat. 1. Hot air sterilization: It is intended for glassware. The apparatus is an oven which is operated at 160°C for 2 hours. 2. Incineration: It is used for heating inoculating loop, and destruction of carcasses, infected laboratory animals, and other infected materials. II. LOW TEMPERATURES: Psychrophiles can grow at 0°C. Subzero temperatures inhibit the metabolism of microorganisms. Low temperatures are used to preserve materials. Lyophilization is a freeze-dried technique used to preserve biological specimens. Dry ice chest: -50° to -70°C Liquid nitrogen: -196°C Revco: -60° to -90°C RADIATION Electromagnetic radiation is energy in the form of electromagnetic waves. It is classified according to its wavelength. Radio waves have the longest wavelength, and cosmic rays have the shortest (Fig. 11.7, p. 340). The energy content is inversely proportional to the wavelength: the shortest the wavelength, the greater the energy content. Some forms of electromagnetic radiation can ionize molecules. 1. Ionizing Radiation: (Fig. 11.8, p.341) The ionizing radiation includes alpha, beta, gamma, X-rays, cathode rays, and high-energy protons and neutrons. They cause ionization of molecules. Water molecules are split into hydroxyl radicals (OH-), electrons, and hydrogen ions (H+). The radicals are highly destructive to DNA , proteins and other molecules. Ionizing radiation is able to penetrate the interiors of packages. Gamma rays are less expensive than X-rays. Cobalt-60 (60Co) emits gamma rays. The emission of gamma rays cannot be turned on or off like an X-ray machine. Sensitivity to ionizing radiation of the cells varies inversely with the size of their DNA volume. Ionizing radiation is used to sterilize packaged meats, plastic hypodermic syringes, and suture. It is also used for the radiopasteurization of fruits, seafood, eggs, milk, poultry products, and stored grains. Radiation sterilization is used to produce vaccines.

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2. Microwave Irradiation: Microwaves are a form of energy which generates heat when they interact with water or oil. The waves cause either negatively charged particles (ions) to accelerate and collide with other molecules or cause dipoles (one pair of electric poles with different charges separated by a short distance) to attempt to rotate and line up with the rapidly alternating electrical field. The usable and safe frequencies range from 915 to 2,450 megahertz (MHz) (a hertz is a frequency unit of one cycle per second). Dry spores or lyophilized cultures are not affected by this treatment. 3. Nonionizing Radiation: Ultraviolet (UV) radiation has a wavelength of 136 to 400 nanometers (nm). The greatest bactericidal activity is around 260 nm. UV light can cause deletion, and the formation of dimers (two adjacent pyrimidines on the same DNA strand become bonded together) (Fig. 11.10, p. 343). Most bacteria are able to repair the DNA damage (formation of pyrimidine dimer) in the presence of light. This is known as photoreactivation. Some bacteria carry out dark reactivation in which damaged DNA is repaired in the absence of light. UV light is often used to reduce the number of microorganisms on the surfaces of hospital operating rooms, and in aseptic rooms. SONICATION: Sound vibrations at high frequency (9 to 500 kilocycles/second) are highly destructive to bacteria. Cells can be ruptured by high frequency sound waves. The high frequency sound waves induce pressure changes and minute, bubble-like cavities in the liquid, a phenomenon called cavitation. The short-lived bubbles swell and collapse, creating intense points of turbulence that can stress and burst cells in the vicinity (Fig. 11.12, p. 344). FILTRATION: Certain materials such as serum, plasma, enzyme solutions, bacterial toxins, cell extracts, ascitic fluid, and sugar solutions cannot tolerate heat sterilization without deterioration. They are filtered through either a Seitz filter, Berkefeld filter or Chamberland-Pasteur filter. They are all made up of a mixture. of diatomaceous earth, asbestos, plaster of Paris, and water. The fritted or sintered glass filters which are made up of finely ground glass particles have also been used. All these filters have been replaced by highly versatile, paper-thin membrane filters made from cellulose acetate and other related materials. Filtration does not remove viruses from serum, plasma or ascites fluid. 1. Membrane Filters: Membrane filters are thin disks (about 150 µm thick) of cellulose esters. They are used for separating microorganisms, and for collecting microbes from air and water which may contain very few microbes. 2. High-Efficiency Particulate Air (HEPA) Filters: CDC (Centers for Disease Control) established four biosafety levels for laboratories that work with biological materials. Level 1 requires only standard, open-bench laboratory techniques, and level 4 requires maximum containment. Special biological safety cabinet has an open front through which air is drawn in and away from the worker. The air exits through a highefficiency particulate air (HEPA) filter which is made of cellulose acetate pleated around aluminum foil. It filters out 99% of the particulate matter from the air.

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DESICCATION Drying of vegetative cells stops their metabolic activity, and leads to the decline of the population. Farmers have used this method for centuries to preserve grains. How long microbes can survive depends on the following factors: 1. The types of organisms. 2. The types of material to be dried. 3. The completeness of drying process. 4. The physical conditions (such as light, temperature, and humidity) in which the material is kept. Gram-negative cocci such as Neisseria gonorrhoeae and N. meningitidis are very susceptible to desiccation. They are killed within minutes of drying. Gram-positive bacteria are more resistant. The endospores of bacteria may survive indefinitely. CHEMICAL AGENTS IN MICROBIAL CONTROL There are approximately 10,000 antimicrobial agents of which about 1,000 of them are routinely used. They may occur in liquid, gaseous or solid state. If the liquid contains water it is termed aqueous; and if it contains alcohol or water-alcohol, it is called a tincture. CHOOSING A MICROBICIDAL CHEMICAL The desirable qualities of a germicide are: 1. Antimicrobial activity. 2. Solubility. 3. Broad-spectrum. 4, Penetration ability and stability. 5. Resistance to inactivation by organic matter. 6. Noncorossive and nonstaining properties. 7. Sanitizing and deodorizing properties 8. Inexpensive and availability. FACTORS THAT Al-l-ECT THE GERMICIDAL ACTIVITY OF CHEMICALS The factors include the nature of microorganisms, the nature of the material to be treated, the degree of contamination, the time of exposure and the concentration of the chemicals (Table 11.6, p. 348). GERMICIDAL CATEGORIES ACCORDING TO CHEMICAL GROUP (TABLE 11.5, P. 347) The following table presents the chemical methods for disinfection and antisepsis. Group

Halogens: Iodine, chlorine, Ca or Na hypochlorite: Ca(OC1)2, NaOCl; chloramines,

Mode of Action

1. Oxidize proteins & inactivate enzymes 2. Disrupt cell membrane

Uses

Purification of water, dairy sanitation, restaurant sanitation

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hydrogen peroxide, halozone (parasulfone dichloramidobenzoic acid) Chlorine and its compounds are antimicrobial due to the formation of nascent oxygen (0) which is a powerful oxidizing agent that can damage cellular substances. C12 + H2O ---------- > HCl + HC1O Hypochlorous acid HClO ----------------- > HCl + 0 (Nascent oxygen) Chlorine can also combine with proteins to destroy their biological activity. Phenol (carbolic 1. Denature proteins acid) and related 2. Damage cell membrane compounds: cresol, hexachlorophene, lysol, phisoderm, pHisohex

Disinfection of laboratory equipment, instruments, bench tops, toilets

Alcohols: ethyl, methyl, propyl, butyl, amyl, octyl

1. Denature proteins 2. Dissolve lipids 3. Dehydration 4. Destroys cell membrane

Skin disinfection; kill vegetative cells, little effect on spores

Detergents: Soaps,

1. Mechanical removal 2. Destroy cell membrane 3.Increase permeability 4. Denature proteins

Quaternary ammonium, 1. Disturb metabolic process compounds: ceepryn, 2. Denature proteins zephiran, phemerol 3. Disrupt cell membrane

Cleansing, mechanical removal and bactericidal action Used as skin antiseptics; as sanitizing agents in eating and drinking establishments, dairy and food processing plants

There are three major groups of detergents: 1. Nonionic detergents: They do not ionize when dissolved in water. They are not antimicrobial. 2. Anionic detergents: The anionic portion of the molecule determines the detergent (wetting) property. 3. Cationic detergents: The cationic portion of the molecule determines the detergent property. They are the most antimicrobial detergents of which quaternary ammonium compounds are the most widely used. Heavy metal compounds: Mercuric bichloride (HgC12), merthiolate, merbromin (mercurochrome), nitromersol (Metaphen),

silver nitrate (AgNO3), copper sulfate (CuSO4)

1. Metallic ions precipitate enzymes or proteins

Laboratory disinfectant, skin antisepsis, preservation of biologicals; 1% AgNO3 is used in antisepsis of throat and eyes (to prevent ophthalmia neonatorum)

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Aldehydes: 1. React with enzymes glutaraldehyde (2%), & nucleic acids formalin (37-40% 2. Denature proteins aqueous formaldehyde, HCHO) Gaseous chemoInactivate enzymes sterilizers: or proteins ethylene oxide, chlorine, sulfur dioxide formaldehyde, methyl bromide Dyes: crystal violet ---gentian violet 1 malachite green acriflavine --------- 2 proflavine --------- 2

86 Disinfection of lensed instruments, sharp instruments, inhalation equipment, thermometers, rubber or plastic items Used for sterilizing heat- or moisturesensitive materials such as blankets and pillows

1. Interfere with cellular Used in selective media; treatment of oxidation processes of burns and wounds Gram-positive bacteria 2. Inhibit Gram-negative bacteria

Acids & Alkalies: 1. Ions combine with boric, nitric, cellular constituents salicylic, 2. Destroy cell wall & undecylinic acids, cell membrane KOH, NaOH, calcium oxide (lime)

Preservation, mild antisepsis; dishwashing & dairy sanitation

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Chapter 12 DRUGS, MICROBES, HOST--THE ELEMENTS OF CHEMOTHERAPY PRINCIPLES OF ANTIMICROBIAL THERAPY The use of drugs to control infectious diseases has significantly increase the lifespan and health of humans. Unfortunately, the diseases have not been eradicated, and the mortality rates caused by infectious diseases are as high as before. THE TERMINOLOGY OF CHEMOTHERAPY Prophylaxis: a process that prevents infection or disease. Chemotherapeutic drug: chemical used in treatment of disease. Antimicrobial chemotherapy: treatment of disease by using antimicrobial drugs. Antibiotics: the natural metabolites produced by microorganisms that inhibit or kill other microorganisms. Narrow-spectrum agents: chemicals that are effective against a limited number of microorganisms. Examples: Bacitracin inhibits certain grampositive bacteria, and griseofulvin is effective against fungal infections. Broad-spectrum agents: chemicals that are effective against a wide range of microorganisms. Example: Tetracyclines are effective against gram-positive and gramnegative bacteria, rickettsias and mycoplasmas. DRUGS, MICROBE, HOST--SOME BASIC INTERACTIONS. The drugs administrated into the patients should kill the pathogens without harming the host cells. The stages of chemotherapy are as follows: (1) The drug is administered to the host via the following routes: per os: by oral (given by mouth) intravenous inoculation: into a vein intramuscular inoculation: into the muscle topical: on the skin surface subcutaneous inoculation: under the skin intracutaneous inoculation: into the skin intraperitoneal inoculation: into the abdominal cavity intrapleural inoculation: into the pleural cavity intracerebral inoculation: into the brain subdural inoculation: beneath the dura mater of the brain or spinal cord per rectum: given by rectum parenteral inoculation: inoculation into the body other than the intestinal tract (2) The drug is dissolved in body fluids. (3) The drug is delivered to the infected area. (4) The drug destroys or inhibits the pathogen. (5) The drug is eventually excreted or broken down. Table 12.1 (p. 361) presents the characteristics of the ideal antimicrobial drugs.

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IDEAL QUALITIES OF A CHEMOTHERAPEUTIC AGENT An ideal chemotherapeutic agent should have the following qualities: 1. Effective against many kinds of pathogens. 2. Prevent the development of antibiotic-resistant strains. 3. Should produce no side effects in the patients. 4. Should not destroy the normal flora. 5. Should be chemically stable, and not inactivated by stomach acid or blood proteins. 6. Should be highly soluble in body fluids so that it can remain active. 7. Must be able to reach the tissues or blood in sufficient concentration to kill the pathogens, and should have no harmful effects to the host. THE ORIGINS OF ANTIMICROBIAL DRUGS Antibiotics are predominantly produced by bacteria in the genera Streptomyces and Bacillus, and molds in the genera Penicillium and Cephalosporium. Semisynthetic and synthetic drugs have been made by chemists. CHARACTERISTIC INTERACTIONS BETWEEN DRUG AND MICROBE Most drugs interfere with the function of enzymes required to synthesize macromolecules or destroy cellular structures. A preferred drug should have selective toxicity which produces adverse effects on pathogens but not the host cell. Penicillin is a good example. It prevents cell wall synthesis in bacteria. But it has no effect on human cells because they do not have a cell wall. Amphotericin B is very toxic to human cells because it destroys the cell membrane which is found in both human cells and bacteria. MECHANISMS OF DRUG ACTION (MODES OF ACTION OF DRUGS) 1.

Inhibition of cell wall synthesis: (Fig. 12.3, p. 365) Some antibiotics exert their effect by inhibition of cell wall synthesis. No cross-linking between peptidoglycan chains can be formed (Fig. 12.4, p. 365). A bacterial cell without cross-links between the peptidoglycan in the cell wall will easily rupture and die. Moreover, the antibiotics cause the loss of the natural inhibitors in the cell wall, and allow the degradative enzymes which are also present in the cell wall to break down the peptidoglycan. Examples: Bacitracin, cephalosporins, penicillins, vancomycin, ristocetin, cycloserine, monobactams and fosfomycin.

2.

Inhibition of nucleic acid synthesis: Interference with the nucleic acids (DNA and RNA) synthesis will lead to the death of the organism. Examples: Actinomycin D, griseofulvin, idoxuridine, rifampin and sulfonamides (sulfa drugs). All sulfonamides have the same core structure with various groups attached to the core. The core structure is similar to para-aminobenzoic acid (PABA) (Fig. 12.5, p. 366). PABA is used as a substrate by bacteria to synthesize a coenzyme called tetrahydrofolic acid (THFA) which is essential to make certain amino acids, purines and pyrimidine (Fig. 12.6, p. 366). Sulfonamides compete with PABA for the active site on the enzyme dihydropteroate synthetase in a type of enzyme

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3.

Inhibition of protein synthesis: Most antibiotics exert their effect by inhibition of protein synthesis. Various stages of protein synthesis are attacked by different antibiotics (Fig. 12.7, p. 367). A cell cannot function and will eventually die if it cannot synthesize proteins. Examples: Chloramphenicol, gentamicin, kanamycin, neomycin, streptomycin, tetracycline, chlotetracycline, oxytetracycline, erythromycin, fucidin, lincomycin, clindamycin, and cycloheximide.

4.

Damage to the cytoplasmic membrane: Some antibiotics kill the bacteria by directly destroying the cell membrane or by increasing the permeability of the cell membrane. A cell cannot survive if important molecules leak out of the cell. Polymyxins destroy the phospholipids of cytoplasmic membrane (Fig. 12.8, p. 367). Nystatin and amphotericin B destroy the sterols of the membrane, and are effective against fungi and animal cells that contain sterols in the membrane, but not against most bacteria which have no sterols in their membranes, Examples: Nystatin, Amphotericin B, vancomycin, colistin, and polymyxins.

5.

Inhibition of metabolism: Certain antibiotics inhibit the formation of a specific metabolic product Examples: Isoniazid (isonicotinic hydrazide, INH), para-aminosalicylic acid (PAS), nitrofurans, sulfonamides and trimethoprim.

THE ACQUISITION OF DRUG RESISTANCE Drug resistance is an adaptive response in microorganisms so that they can tolerate an amount of drug that would normally be inhibitory. Genetic versatility enables microorganisms to develop mechanisms to circumvent or inactivate antimicrobics. How Does Drug Resistance Develop? The appearance of antibiotic resistance is due to mutation. The plasmids containing genetic codes for drug resistance (resistance factors or R factors) can be transferred through conjugation, transformation or transduction. Transposons may also contain the genetic codes. They can jump from one plasmid to another or from a plasmid to the chromosome. The ease of transfer even between species accounts for the rapid proliferation of drug-resistance species (Fig. 12.9, p. 368). Specific Mechanisms of Drug Resistance: The mechanisms that are responsible for antibiotic resistance are summarized as follows: 1. Some bacteria may produce enzymes that destroy or modify the structure of the antibiotics. 2. The antibiotics may not be able to pass through an outer membrane (envelope) to exert their effects inside the cells. 3. There may be a change in the number of receptor sites for the uptake of the drug. 4. The bacterium may use an alternative biochemical pathway if its main pathway is inhibited by the antibiotic. 5. The bacterium may have enzymes, ribosomes or other cell components that are not affected by the antibiotic.

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6. The bacteria may simply lapse into dormancy or convert to an L-form which is not affected by the antibiotics such as penicillins. Drug Inactivation Mechanisms: Penicillins can be inactivated by beta-lactamases (also called penicillinases). The enzymes hydrolyze the beta-lactam ring structure of some penicillins and cephalosporins, and making them inactive (Fig. 12.10a, p. 368). Alternate drugs have to be used for penicillinase-producing strains of Staphylococcus aureus and Neisseria gonorrhoeae (PPNG). Chloramphenicol can be inactivated by the addition of an acetyl group by an enzyme. Other antibiotics can be inactivated by the addition of phosphate groups or adenyl groups. Decreased Permeability to a Drug: The outer membrane (envelope) of gram-negative bacteria effectively blocks the entrance of some penicillin. Proteins encoded by the plasmid in tetracycline-resistant bacteria pump the drug out of the cell. Resistance to the aminoglycoside antibiotics (streptomycin and neomycin) is due to the loss of the capacity to transport the drug intracellularly. Change of Drug Receptors. Resistance to rifampin and streptomycin is due to a change in the receptor proteins on the cell membrane. The drugs are no longer able to bind to the receptors. Erythromycin and clindamycin resistance is due to the alteration on the 50S ribosomal binding site. Penicillin resistance in Streptococcus pneumoniae and methicillin resistance in Staphylococcus aureus are due to an alteration in the binding proteins in the cell wall. Fungi become resistant to certain antifungal drugs by decreasing the synthesis of ergosterol which is the principal receptor for the drugs. Changes in Metabolic Patterns: Bacteria may develop an alternative pathway or enzyme to circumvent their usual metabolic pathway which can be blocked by the drugs (Fig. 12.10b, p. 368). Natural Selection and Drug Resistance: Drug resistance arises through mutation. The cells with resistant genes are already out there in nature. But the number of such cells remains low as the cells have no selective advantage. When drug is applied, the cells that do not possess the genes are wept out. Only the ones with the genes will survive. The cells will proliferate, giving rise to a drug-resistant population (Fig. 12.11, p. 369). SURVEY OF MAJOR ANTIMICROBIAL DRUG GROUPS There are about 260 antimicrobial drugs classified in 20 drug families. Table 12.2 (p. 371372) summarizes some drugs used for the treatment of some major infectious diseases. Based on the range of antimicrobial activity, antibiotics can be divided into broadspectrum and narrow-spectrum antibiotics. A broad-spectrum antibiotic is effective against a wide variety of microorganisms. A narrow-spectrum antibiotic is effective against only a few species or a single bacterial group. The following table (next page) lists examples of broad-spectrum and narrowspectrum antibiotics.

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Narrow-Spectrum Antibiotics

Amphotericin B (Fungizone) Bacitracin Colistin Dihydrostreptomycin Gentamicin Griseofulvin Kanamycin Lincomycin Methicillin Nafcillin Novobiocin Nystatin Oxacillin Penicillin Polymyxins Streptomycin Spectinomycin Vancomycin Ampicillin Cephalosporins Cephalothin Chloramphenicol Clindamycin Erythromycin Imipenem Kanamycin Neomycin Paromomycin Rifampin Tetracyclines Tobramycin

ANTIBACTERIAL DRUGS Penicillin and Its Relatives: All penicillins consist of three parts: a thiazolidine ring, a beta-lactam ring, and a side chain (R group) (Fig. 12.12, p. 366). They are predominantly produced by Penicillium chrysogenum. Subgroups and Uses of Penicillins: Table 12.3 (p. 366) shows the characteristics of some penicillins. Penicillins G and V are used to treat infections caused by gram-positive cocci (streptococci, pneumococci) and some gram-positive bacteria (meningococci and spirochetes). Penicillin V is used as a prophylaxis for children with sickle-cell anemia who are threatened by systemic pneumonococcal infections. Methicillin, nafcillin and cloxacillin are used to treat infections caused by penicillinaseproducing bacteria. Mezlocillin and azlocillin have very broad spectrum of action. They can substitute the use of drug combination. One international unit of penicillin is defined as the strength of an antibiotic which is equivalent to the antimicrobial activity exerted by 0.6 pg (0.0000006 g; 1 mg contains 1,667 units) of pure sodium penicillin G. One unit is sufficient to inhibit the growth of Bacillus subtilis in 20 to 50 ml of broth. The Cephalosporin Group of Drugs: Cephalosporins are first isolated from Acremonium acremonium (previously called Cephalosporium acremonium). They are similar to penicillins in having a beta-lactam ring structure (Fig. 12.13, p. 374), and the mode of action.

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Subgroups and Uses of Cephalosporins: Cephalosporins are broad-spectrum, resistant to penicillinases, and cause fewer allergic reactions than penicillins. Most are poorly absorbed through the intestine and must be administered parenterally (a route other than the intestinal tract). Cephalosporins are grouped as first-generation (cephalothin, cefazolin), second-generation (cefaclor, cefonacid, cefamandole, cefoxitin, and cefuroxime), and third-generation cephalosporins (cephalexin (Keflex), cefotaxime, cefoperazone, and ceftriaxone). The secondand third-generation cephalosporins have a broader spectrum of activity, and are more resistant to enzymatic inactivation. Other Beta-Lactam Antibiotics: Imipenem is broad-spectrum; effective against gram-positive and gram-negative aerobic and anaerobic pathogens. It has low toxicity and can be taken by mouth. Aztreonam is isolated from the purple pigmented bacterium Chromobacterium violaceium. It is a narrow-spectrum drug used to treat pneumonia, septicemia and urinary infections caused by Pseudomonas and other gram-negative bacteria. The Aminoglycoside Drugs: They contain a ring structure called aminocyclitol (6-carbon) ring and amino sugars (Fig. 12.14, p. 374). They are produced by Streptomyces (Fig. 12.15, p. 374) and Micromonospora. Subgroups and Uses of Aminoglycosides: Most are broad-spectrum in action by inhibiting protein synthesis. They are used to treat infections caused by aerobic gram-negative rods and some gram-positive bacteria. Streptomycin is produced by Streptomyces griseus. It was first isolated by in 1944 by Selman Waksman and his colleagues at Rutgers University. It inhibits many bacteria that are resistant to sulfonamides and penicillin. It is effective against many gramnegative bacteria, and the causative agents of tularemia, plague, and tuberculosis. Streptomycin has a cumulative detrimental effect on the nervous system if it is used for a long periods of time. Gentamicin is less toxic. It is used to treat infections caused by gram-negative bacteria (Escherichia coli, Proteus, Pseudomonas, Salmonella, and Shigella). Neomycin is the most toxic antibiotic in the aminoglycoside group. Since it is poorly absorbed, it is given orally prior to intestinal surgery to help suppress the flora of large intestine. Neomycin is a component of topical ointment. Kanamycin is broad-spectrum and is nephrotoxic and ototoxic (can cause deafness). It must be given parenterally. It has been replaced by tobramycin and amikacin. Spectinomycin is used in one-dose intramuscular treatment of gonorrhea. It is not nephrotoxic or ototoxic. Tetracycline Antibiotics: Tetracyclines have a chemical structure called a naphthalene ring (consists of four rings) attached with different chemical groups (Fig. 12.16, p. 375). They include the natural aureomycin (produced by Streptomyces) and semisynthetic products such as terramycin and tetracycline. They are collectively called tetracyclines. They are broad-spectrum. They bind to ribosomes and block protein synthesis. Subgroups and Uses of Tetracyclines: They include chlortetracycline, oxytetracycline, tetracycline, doxycycline, and minocycline. They are effective against gram-positive bacteria, gram-negative bacteria, the

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organism that causes brucellosis, and the organisms (mycoplasmas, rickettsias, and chlamydias) that cause urinary tract infections. Doxycycline and minocycline have a broader antimicrobial spectrum, and are better absorbed by the body than other tetracyclines. Chloramphenicol: It was first isolated in 1940s from Streptomyces venezuelae. Because of its simple structure--with a nitrobenzene ring (Fig. 12.16b, p. 375), it can be economically synthesized. It is a broad-spectrum antibiotic. It causes aplastic anemia, a fatal condition in which bone marrow fails to produce new blood cells. It is used for treatment of typhoid fever, brain abscesses, eye infections, meningitis, and other infections caused by antibiotic resistant bacteria. The Macrolides: The macrolides contain a large lactone ring linked with amino sugars (Fig. 12.16c p. 368). The examples are erythromycin, clarithromycin and azithromycin. Erythromycin is produced by Streptomyces erythreus originally isolated from soil collected in Philippines. It is broad-spectrum, and has low toxicity. It inhibits protein synthesis by binding to the 50S ribosomal subunit.. It is effective against gram-positive and gramnegative bacteria, and pathogenic spirochetes. It is not destroyed by penicillinase, and is often used as an alternative to penicillin. Clindamycin is synthesized from lincomycin. It is broad-spectrum. It causes adverse reaction. It is used for treatment of infections of the intestines caused by Bacteroides and Clostridium; penicillinresistant staphylococci, and acne. Vancomycin is a glycopeptide antibiotic. It is narrow-spectrum and is toxic. It is used to treat infections caused by Clostridium and Enterococcus faecalis (causes endocarditis). Rifampin is synthesized from rifamycin produced by Streptomyces. It is too large to pass through the cell envelope. It blocks transcription and inhibits cell growth. It is used to treat tuberculosis, leprosy, gonorrhea, and infections caused by Legionella, Brucella and Staphylococcus. Overdose of rifampin produces the "red-man syndrome" due to the excretion of the pigmented metabolic products which cause the yellow-orange or orange-red to red discoloration of the skin. The pigment also appears in tears and urine. The Bacillus Antibiotics: Bacitracin and Polymyxin The Polypeptide Antibiotics: Bacitracin is produced by Bacillus subtilis. It is a narrow-spectrum antibiotic used for topical application. It is a major ingredient in an antibiotic ointment called Neosporin for skin infections caused by streptococci and staphylococci. It is usually combined with neomycin (an aminoglycoside) and polymyxin. Colistin (polymyxin E) is produced by Bacillus colistinus and other polymyxins are produced by Bacillus polymyxa. Polymyxins have a circular peptide chain attached with a fatty acid which contributes to their detergent activity. Polymyxins (A, B, C, D and E) are effective against gram-negative bacteria such as Pseudomonas aeruginosa. Because of their nephrotoxicity and neurotoxicity in the host, they are also used for topical application. The Polyenes: Polyene antibiotics are related to macrolide'antibiotics. They also contain a large lactone ring with a conjugated double-bond system. They interact with sterols in the plasma membrane and alter the permeability of the membrane, causing leakage of small molecules from the cells. The two wellknown examples are nystatin and amphotericin B (fungizone). Nystatin, produced by

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AGENTS TO TREAT FUNGAL INFECTIONS Amphotericin B (Fungizone) was isolated from Streptomyces. It is the most effective of all antifungals. It is effective against skin and mucous membrane infections caused by Candida albicans, and systemic fungal infections. Nystatin was isolated from Streptomyces. It is used to treat subcutaneous or systemic fungal infections or ringworm infections. Griseofulvin is produced by Penicillium. It is used to treat athlete's foot. It is nephrotoxic and is used in only the most extreme cases. The axoles are broad-spectrum antifungal agents with a complex ringed structure. They include: Ketoconazole is one of the most effective drugs. It is used orally and topically for cutaneous mycoses, vaginal and oral candidiasis, and some systemic mycoses. Clotrimazole and miconaxole are used to treat skin, mouth and vagina infections. Flucytosine is an analog of cytosine that was first developed for tumor therapy. It is rapidly absorbed. It is used to treat Cryptococcus meningitis and candidiasis. It is used in combination with amphotericin B to treat the resistant fungi. ANTIPARASITIC CHEMOTHERAPY Antimalarial Drugs: Quinine and Its Relatives Quinine is a malaria drug extracted from the bark of the cinchona tree in South America. It was replaced by the synthesized quinolines (mainly chloroquine and primaquine) after World War II. Primaquine is effective against the liver phase of infection, and chloroquine is effective against the infection of red blood cells. They are used in combination as prophylaxis and cure. Due to the appearance of resistant strains of Plasmodium, quinine has made a comeback. Chemotherapy for Other Protozoan Infections: Metronidazole (Flagyl) is effective against Entamoeba histolytica (causes amoebic dysentery, Giardia lamblia (causes giardiasis), and Trichornonas vaginalis (trichimoniasis). Synthetic Antiprotozoan Agents: Chemotherapeutic Agent

Disease

Causative Agent

Chloroquine Primaquine Quinine

Malaria

Plasmodium falciparum Plasmodium vivax Plasmodium ovale Plasmodium malariae

Pentamidine; suramin

African sleeping sickness (African trypanosomiasis)

Trypanosoma gambiense Trypanosoma brucei

Nitrofurfurylidine derivative

Chagas's disease (American Trypanosoma cruzi trypanosomiasis, American sleeping sickness)

Antimony sodium gluconate; pentamidine

Leishmaniasis

Leishmania donovani Leishmania brasiliensis

Quinacrine or metronidazole

Giardiasis (giardial dysentery)

Giardia lamblia

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Metronidazole and diiodohydroxyquin

Amoebiasis (amoebic dysentery)

Entamoeba histolytica

Metronidazole

Trichomoniasis

Trichomonas vaginalis

Pyrimethamine plus trisulfapyrimidines

Toxoplasmosis

Toxoplasma gondii

Trimethoprim and sulfamethoxazole Pneumocystosis (pneumonias)

Pneumocystis carinii

Antihelminthic Drug Therapy: Mebendazole and thiabendazole are broad-spectrum drugs against roundworms and tapeworms. They inhibit the function of the microtubules of adult worms, and thus interfere the glucose utilization and disabling them. They also kill eggs and larvae. Pyrantel and piperazine paralyze the muscles of roundworms. Niclosamide destroys the scolex and the proglottids of tapeworms. The worms lose their grip on the intestinal wall and are expelled from the body. Praziquantel is used to treat tapeworm and fluke infections. ANTIVIRAL CHEMOTHERAPEUTIC AGENTS Very few chemicals are available for the treatment of viral diseases. The reason is that virus multiplies within the host cells by using the host cellular machinery. In order to destroy the virus, the drug has to enter the cell and selectively destroy the virus but not the host. Unfortunately, many drugs that inhibit viral replication also inhibit the host-cell metabolism. Most antiviral drugs are structural analogs of purines or pyrimidines. They work by (1) preventing the entrance of viruses, (2) blocking the transcription and translation of viruses, and (3) preventing the maturation of viruses. Acyclovir (also known as acycloguanoside or Zovirax) is an analog of deoxyguanosine (deoxyribose + guanine). It blocks DNA synthesis and is effective against herpesvirus types 1 and 2, varicella-zoster virus, and Epstein-Barr virus. Famcicloviris, related to acyclovir, is used to treat shingles and chickenpox caused by the varicellazoster virus. Idoxuridine: It is an analog of thymidine (thymine + deoxyribose). It is used only for treatment of eye infections caused by herpesviruses. Vidarabine (ara-A) has replaced idoxuridine. It is an analog of deoxyadenosine (deoxyribose + adenine). It is effective against DNA viruses (herpes simplex and varicella-zoster viruses). Ribavirin, an analog of guanine, is used in aerosol form to treat infections caused by the respiratory syncytial virus (RSV) in infants. Azidothymidine (also known as AZT; zodovudine, ZDV or Retrovir; or 3'-azido-2', 3dideoxythymidine): AZT is an analog of thymidine. It is also known as reverse transcriptase inhibitor. It has N3 connected to the 3' carbon of the sugar molecule. Once AZT has been incorporated, no further nucleotides can be added to the 3' end. It thus prevents the replication of HIV by halting DNA synthesis. It has no effect on human cells because human cells do not use

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reverse transcriptase to synthesize DNA as the virus does. Among its side effects are bone marrow suppression which leads to anemia and decreased numbers of platelets and granulocytes. Other approved anti-AIDS drugs that have similar action are didanosine (ddl), zalcitabine (ddC), stavudine (d4T) and epivir or lamivudine (3TC) . Protease inhibitors prevent protease of HIV from cutting its long chain of parental viral protein into its protein components (p 18, p24, p41, gp 120) and enzymes so as to assemble into mature and fully infectious viral particles. The new copies of HIV can still be made. But they are defective and cannot infect other cells. They include ritonavir (norvir), indinavir, saquinavir, nelfmavir and 141 W94 (VX-478). Amantadine (Symmetrel) and its relative, rimantidine, are used to treat infections caused by influenza A virus only. They inhibit the uncoating of the virus. Interferon was discovered in 1957 by Alick Isaacs and Jean Lindenmann of the National Institute for Medical Research in London. It is a complex of sugar and protein called glycoprotein. There are three families of interferon, produced by leukocyte (alpha interferon), fibroblasts, and connective tissues (beta interferon), and T lymphocytes (gamma interferon). It is now mass produced by genetic engineering technique. It inhibits viral replication. It is used to treat infections caused by herpesviruses, influenza virus, papillomaviruses (warts), hepatitis C, AIDS, hairy-cell leukemia (caused by HTLV-II), Kaposi's sarcoma, bone cancer, cervical cancer, and lymphomas. The side effects of interferon include fever, chills, loss of appetite, nausea, vomiting, headache, muscle and joint pain, temporary loss of blood cell production, seizures and cardiac complications. CHARACTERISTICS OF HOST/DRUG REACTIONS About 5% of people taking antimicrobial drugs experience serious reactions. Refer to Table 12.4 (p. 380) for the adverse toxic reactions. TOXICITY TO ORGANS The hepatotoxic drugs cause enzymatic abnormalities, fatty liver deposits, hepatitis and liver failure. The nephrotoxic drugs interfere with the filtration abilities of nephron tubules. Sulfonamides can crystallize in kidney pelvis and form kidney stones. Some drugs cause diarrhea by irritating the intestinal linings and replacement of normal flora. Drugs used to treat parasitic infections may cause irregular heart beats and cardiac arrest. Chloramphenicol can cause either a reversible or permanent (fatal) anemia. Some drugs can damage red blood cells and platelets. The neurotoxic drugs (aminoglycosides) damage nerves (especially the 8th cranial nerve), causing dizziness, deafness or motor and sensory disturbances, and respiratory failure. Some drugs cause skin allergy or a direct toxic effect, or interact with sunlight to cause photodermatitis. Tetracyclines bind to the enamel of the teeth, and cause a permanent gray to brown discoloration (Fig. 12.19, p. 381). They can pass through the placenta and deposited in the developing fetal bones and teeth. ALLERGIC RESPONSES TO DRUGS Allergy is the most frequent drug reaction. The reaction is prompted by the drug itself or its metabolite. For example, allergy to penicillin is caused by its breakdown product called benzylpenicilloyl. Penicillin reaction accounts for the greatest number of drug allergies followed by the sulfonamides. A person becomes sensitized to the drug after the first exposure. But, upon the second exposure, the following reactions may occur: skin rash (hives), respiratory inflammation, and rarely anaphylaxis which can be fatal.

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SUPPRESSION AND ALTERATION OF THE MICROFLORA BY ANTIMICROBICS Superinfection refers to the overgrowth of opportunistic microbial populations or contaminants after the elimination of beneficial normal flora by drugs. For example, the treatment of urinary tract infection caused by Escherichia coli kills the organism, and also the beneficial lactobacilli which maintain a low pH in the vagina. In the absence of lactobacilli, a yeast called Candida albicans proliferates and causes an infection. It also causes similar superinfections of the oropharynx (thrush) and the large intestine. Oral therapy with tetracyclines, clindamycin and broad-spectrum penicillins and cephalosporins causes antibiotic-associated colitis (pseudomembranous colitis). It is due to the overgrowth of a spore-former called Clostridium difficile which produces toxins that cause diarrhea, fever, and abdominal pain. CONSIDERATIONS IN SELECTING AN ANTIMICROBIAL DRUG Three factors must be considered in any chemotherapy: (1) the nature of the pathogen, (2) the susceptibility of the pathogen, and (3) the medical condition of the patient. IDENTIFYING THE AGENT A doctor often begins the initial therapy based on the information on the direct microscopic examination of a Gram-stained clinical specimen which may give the possible identity of a pathogen. The choice of drug is based on past experience. For example, if a sore throat is suspected to be caused by Streptococcus pyogens, penicillin is often prescribed because it is effective. If a pathogen is not or cannot be isolated, epidemiological statistics are used to predict the most likely pathogen. For example, Haemophilus influenzae is responsible for most meningitis cases in children. It is followed by Streptococcus pneumoniae and Neisseria meningitidis. TESTING FOR THE DRUG SUSCEPTIBILITY OF MICROORGANISMS ASSAY OF ANTIBIOTICS Chemical Assay: Chemical assay is used to determine the antimicrobial activity of an antibiotic in solid form. It is expressed in micrograms of the antibiotic per milligram (p.g/mg) of the specimen. It requires less time to complete, and is less sensitive than the biological assay. Biological Assay: Biological assay is used to measure the amount of antimicrobial activity present in an antibiotic-containing specimen against living microorganisms. It compares the antimicrobial activity of the test antibiotic with the standard antibiotic under standardized conditions. The unit of measurement for most antibiotics follows the international agreement. The biological assay is performed by either tube-dilution technique or dish-plate technique. The amount of antibiotic in a specimen is determined by comparing its activity with that of a standard known concentration of antibiotic. In the example illustrated in Fig. 12.22 (p. 385), the minimum inhibitory concentration of the unknown antibiotic is 4.tg/m1. MICROBIAL SUSCEPTIBILITY TO CHEMOTHERAPEUTIC AGENTS

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The disk-plate technique and tube-dilution technique are used to determine the susceptibility of pathogens to chemotherapeutic agents. Disk-Plate Technique: The Kirby-Bauer method is a highly standardized technique recommended by the Food and Drug Administration (FDA). A test organism is inoculated on an agar plate, and small paper disks impregnated with known amounts of different kinds of antibiotics are placed on the agar. After incubation, the plate is observed for the zone of inhibition which is measured to determine susceptibility (Fig. 12.21, p. 384). The profile of anitmicrobic sensitivity (also called antibiogram) provides data for drug selection. This method is less effective for anaerobic, highly fastidious, or slow-growing bacteria. Tube-Dilution Technique: Serial dilutions of an antibiotic are made in test tubes, and a test microorganism is inoculated into the tubes which are then incubated for 18-24 hours. The lowest concentration of antibiotic that prevents a visible growth is called the minimum inhibitory concentration (MIC) of the antibiotic. It is expressed in p g/ml. The antibiotics that have the lowest MIC values have the highest antimicrobial activity. In most clinical laboratories, the procedures are automated (Fig. 12.22b, p. 385). Applying the Results of Drug Susceptibility The results of antibiotic sensitivity testing provide information to a physician the drug of choice. Once the therapy has started, observation of the patient's clinical response is made. The in vitro activity of the drug may not correlate with its in vivo effect. The failure of a drug therapy may be due to: (1) the inability of the drug to get to the body part, (2) the presence of a few resistant cells which did not appear in the sensitivity testing, and (3) the infection may be caused by more than one pathogen which may be resistant to the drug. In the event of a drug failure, a different drug, combined therapy or different route of administration must be used. MEDICAL CONSIDERATIONS IN ANTI-INFECTIVE CHEMOTHERAPY It is better to use the narrow-spectrum drug which has been proved to be effective to avoid superinfections and other adverse reactions. A drug with a high selective toxicity for the pathogen and low human toxicity should be chosen. The ratio of the toxic dose to the effective dose is called the therapeutic index. Maximal tolerated dose (or minimum toxic dose) in the host per kg body wt. Therapeutic index = - --------------------------------------------------------------------Minimal curative dose (or effective microbial lethal dose) per kg body wt. A smaller ratio (therapeutic index) means a greater potential for toxic drug reaction. A higher ratio (therapeutic index) means the drug has a wider margin of safety. For example: a drug with a 10 µg/ml toxic dose Therapeutic index = -----------------------------------------------1.1 = 9 µg/ml (MIC) is riskier than a drug with a: 10 µg/ml toxic dose 10 Therapeutic index = -----------------------------------------------= 1µg/ml (MIC)

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Other considerations include the patient's preexisting medical conditions, age of the patient, the possible interaction with other drugs (the combination of aminoglycosides and cephalosporins increases nephrotoxic effects, antacids reduce the absorption of isoniazid, and the interaction of tetracycline or rifampin with oral contraceptives can abolish the contraceptive's effect), the patient's genetic or metabolic abnormalities, the site of infection, the route of administration, and the cost of the drug. The Art and Science of Choosing an Antimicrobic Drug: The treatment of pneumonia caused by Serratia marcescens in an elderly alcoholic patient complicated by diminished liver and kidney function is the parenteral inoculation of the drugs which do not cause allergy, interaction with alcohol or nephrotoxicity. For a cancer patient with severe systemic Candida tropicalis infection, intravenous inoculation of amphotericin B alone or in combination with flucytosine is the only choice. In a life-threatening situation, a dangerous chemotherapy is the only choice for the survival of the patient. More therapeutic options are available in an otherwise healthy patient. AN ANTIMICROBIAL DRUG DILEMMA The dilemma of using antimicrobials: 1. About half of roughly 200 million prescriptions for antibiotics are inappropriate because the infection is viral in origin. Many drugs are misprescribed as to type, dosage or length of therapy. 2. The overuse of antimicrobials has caused an increase in antimicrobial resistance. 3. The tendency to use a broad-spectrum drug instead of a more specific narrow-spectrum one has led to superinfections and toxic reactions. 4. No antibiotic sensitivity testing is done for the prescribed drug. 5. More expensive drugs are often prescribed. 6. An excess antimicrobial drugs produced in this country is exported to other countries where drugs can be sold on the counters without prescription. This may cause the rise of drugresistant bacteria which can cause epidemics.