General Bacteriology

Chapter 3

General Bacteriology 

The Morphology and Fine Structure of Bacteria

• Bacterial cells are between 0.3 and 5 lm in size. They have three basic forms: cocci, straight rods, and curved or spiral rods. The nucleoid consists of a very thin, long, circular DNA molecular double strand that is not surrounded by a membrane. Among the nonessential genetic structures are the plasmids. The cytoplasmic membrane harbors numerous proteins such as permeases, cell wall synthesis enzymes, sensor proteins, secretion system proteins, and, in aerobic bacteria, respiratory chain enzymes. The membrane is surrounded by the cell wall, the most important element of which is the supporting murein skeleton. The cell wall of Gram-negative bacteria features a porous outer membrane into the outer surface of which the lipopolysaccharide responsible for the pathogenesis of Gram-negative infections is integrated. The cell wall of Gram-positive bacteria does not possess such an outer membrane. Its murein layer is thicker and contains teichoic acids and wall-associated proteins that contribute to the pathogenic process in Gram-positive infections. Many bacteria have capsules made of polysaccharides that protect them from phagocytosis. Attachment pili or fimbriae facilitate adhesion to host cells. Motile bacteria possess flagella. Foreign body infections are caused by bacteria that form a biofilm on inert surfaces. Some bacteria produce spores, dormant forms that are highly resistant to chemical and physical nixie.

Bacterial Forms

• Bacteria differ from other single-cell microorganisms in both their cell structure and size, which varies from 0.3–5 lm. Magnifications of 500– 1000! —close to the resolution limits of light microscopy—are required to obtain useful images of bacteria. Another problem is that the structures of objects the size of bacteria offer little visual contrast. Techniques like phase contrast and dark field microscopy, both of which allow for live cell observation, are used to overcome this difficulty. Chemical-staining techniques are also used, but the prepared specimens are dead.

• Simple staining. In this technique, a single staining substance, e.g., methylene blue, is used. & 

Differential staining

• Two stains with differing affinities to different bacteria are used in differential staining techniques, the most important of which is gram staining. Gram-positive bacteria stain blue-violet, Gram-negative bacteria stain red (see p. 211 for method). Three basic forms are observed in bacteria: spherical, straight rods, and curved rods.

Fine Structures of Bacteria

Nucleoid (Nucleus Equivalent) and Plasmids

• The “cellular nucleus” in prokaryotes consists of a tangle of double-stranded DNA, not surrounded by a membrane and localized in the cytoplasm (Fig. 3.5). In E. coli (and probably in all bacteria), it takes the form of a single circular molecule of DNA. The genome of E. coli comprises 4.63 ! 106 base pairs (bp) that code for 4288 different proteins. The genomic sequence of many bacteria is known.

• The plasmids are nonessential genetic structures. These circular, twisted DNA molecules are 100–1000! smaller than the nucleoid genome structure and reproduce autonomously (Fig. 3.6). The plasmids of human pathogen bacteria often bear important genes determining the phenotype of their cells (resistance genes, virulence genes).

• The cytoplasm contains a large number of solute low- and high-molecular weight substances, RNA and approximately 20 000 ribosomes per cell. Bacteria have 70S ribosomes comprising 30S and 50S subunits. Bacterial ribosomes function as the organelles for protein synthesis. The cytoplasm is also frequently used to store reserve substances (glycogen depots, polymerized metaphosphates, lipids)

The Cytoplasmic Membrane

• This elementary membrane, also known as the plasma membrane, is typical of living cells. It is basically a double layer of phospholipids with numerous proteins integrated into its structure. The most important of these membrane proteins are permeases, enzymes for the biosynthesis of the cell wall, transfer proteins for secretion of extracellular proteins, sensor or signal proteins, and respiratory chain enzymes.

• In electron microscopic images of Gram-positive bacteria, the mesosomes appear as structures bound to the membrane. How they function and what role they play remain to be clarified. They may be no more than artifacts.

Cell Wall

• The tasks of the complex bacterial cell wall are to protect the protoplasts from external oxea, to withstand and maintain the osmotic pressure gradient between the cell interior and the extracellular environment (with internal pressures as high as 500–2000 kPa), to give the cell its outer form and to facilitate communication with its surroundings.

Murein (syn. peptidoglycan)

• The most important structural element of the wall is murein, a netlike polymer material surrounding the entire cell (sacculus). It is made up of polysaccharide chains crosslinked by peptides.

The cell wall of Gram-positive bacteria

• The murein sacculus may consist of as many as 40 layers (15–80 nm thick) and account for as much as

• 30% of the dry mass of the cell wall. The membrane lipoteichoic acids are anchored in the cytoplasmic membrane, whereas the cell wall teichoic acids are covalently coupled to the murein. The physiological role of the teichoic

• acids is not known in detail; possibly they regulate the activity of the autolysins that steer growth and transverse fission processes in the cell. Within the microorganism, teichoic acids can activate the alternative complement pathway and stimulate macrophages to secrete cytokines. Examples of cell wall-associated proteins are protein A, the clumping factor, and the fibronectin-binding protein of Staphylococcus aureus or the M protein of Streptococcus pyogenes. Cell wall anchor regions in these proteins extending far beyond the murein are bound covalently to its peptide components. Cell wall-associated proteins frequently function as pathogenicity determinants (specific adherence; phagocyte protection).

The cell wall of Gram-negative bacteria

•  Here, the murein is only about 2 nm thick and contributes up to 10% of the dry cell wall mass (Fig. 3.11). The outer membrane is the salient structural element. It contains numerous proteins (50% by mass) as well as the medically critical lipopolysaccharide.

Outer membrane proteins

• — Oma (outer membrane protein A) and the murein lipoprotein form a bond between outer membrane and murein. — Porins, proteins that form pores in the outer membrane, allow passage of hydrophilic, low-molecular-weight substances into the periplasmic space. — Outer membrane-associated proteins constitute specific structures that enable bacteria to attach to host cell receptors. — A number of Omps are transport proteins. Examples include the Lamb proteins for maltose transport and Feela for transport of the siderophore ferric (Fe3+) enterochelin in E. coli.

Lipopolysaccharide (LPS. 

• This molecular complex, also known as endotoxin, is comprised of the lipoid A, the core polysaccharide, and the O-specific polysaccharide chain.

Lipoid A is responsible for the toxic effect 

• As a free substance, or bound up in the LPS complex, it stimulates—by binding together with the LPS binding protein (LBP) to the CD14 receptor of macrophages—the formation and secretion of cytokines that determine clinical endotoxin symptomatology. Interleukin 1 (IL-1) and tumor necrosis factor (TNF) induce an increased synthesis of prostaglandin E2 in the hypothalamus, thus setting the “thermostat” in the temperature control center higher, resulting in fever. Other direct and indirect endotoxin effects include granulopoiesis stimulation, aggregation and degeneration of thrombocytes, intravital coagulation due to factor VII activation, a drop in blood pressure, and cachexia. LPS can also activate the alternative complement pathway. Release of large amounts of endotoxincanleadtoseptic (endotoxic) shock. Endotoxinisnotinactivated by vapor sterilization. Therefore,theparentmaterials usedinproductionof parenteral pharmaceuticals must be free of endotoxins (pyrogens).

• The O-specific polysaccharide chain is the so-called O antigen, the fine chemical structure of which results in a large number of antigenic variants useful in bacterial typing (e.g., detailed differentiation of salmonella types)

• L-forms (L = Lister Institute). The L-forms are bacteria with murein defects, e.g., resulting from the effects of beta lactam antibiotics. L-forms are highly unstable when subjected to osmotic influences. They are totally resistant to Beta lactams, which block the biosynthesis of murein. The clinical significance of the L-forms is not clear. They may revert to the normal bacterial form when beta lactam therapy is discontinued, resulting in a relapse.

Capsule

• Many pathogenic bacteria make use of extracellular enzymes to synthesize a polymer that forms a layer around the cell: the capsule. The capsule protects bacterial cells from phagocytosis. The capsule of most bacteria consists of a polysaccharide. The bacteria of a single species can be classified in different capsular serovars (or serotypes) based on the fine chemical structure of this polysaccharide.

Flagella

Flagella give bacteria the ability to move about actively. The flagella (singular flagellum) are made up of a class of linear proteins called flagellins. Flagel late bacteria are described as monostiches, lipoteichoic, or peritrichous, depending on how the flagella are arranged. The basal body traverses the cell wall and cytoplasmic membrane to anchor the flagellum and enables it to whirl about its axis like a propeller. In Enterobacteriaceae, the flagellar antigens are called H antigens. Together with the O antigens, they are used to classify bacteria in serovars.

• late bacteria are described as monostiches, lophobranchs, or peritrichous, depending on how the flagella are arranged. The basal body traverses the cell wall and cytoplasmic membrane to anchor the flagellum and enables it to whir about its axis like a propeller. In Enterobacteriaceae, the flagellar antigens are called H antigens. Together with the O antigens, they are used to classify bacteria in serovars.

Attachment Pili (Fimbriae), Conjugation Pili

• Many Gram-negative bacteria possess thin microfibrils made of proteins (0.1– 1.5 nm thick, 4–8 nm long), the attachment pili. They are anchored in the outer membrane of the cell wall and extend radially from the surface. Using these structures, bacteria are capable of specific attachment to host cell receptors (ligand—receptor, key—keyhole). The conjugation pili (syn. sex pili) in Gram-negative bacteria are required for the process of conjugation and thus for transfer of conjugative plasmids.

Biofilm

• A bacterial biofilm is a structured community of bacterial cells embedded in a self-produced polymer matrix and attached to either an inert surface or living tissue. Such films can develop considerable thickness (mm). The bacteria located deep within such a biofilm structure are effectively isolated from immune system cells, antibodies, and antibiotics. The polymers they secrete are frequently glycosides, from which the term glycocalyx (glycoside cup) for the matrix is derived.

Bacterial Spores

Bacterial spores (endospores) are purely dormant life forms. Them development from bacterial cells in a “vegetative” state does not involve assimilation of additional external nutrients. They are spherical to oval in shape and are characterized by a thick spore wall and a high level of resistance to chemical and physical nixie. Among human pathogen bacteria, only the genera Clostridium and Bacillus produce spores. The heat resistance of these spores is their most important quality from a medical point of view, since heat star

• irisation procedures require very high temperatures to kill them effectively. Potential contributing factors to spore heat resistance include their thick wall structures, the dehydration of the spore, and crosslinking of the proteins by the calcium salt of pyridine-2,6-dicarboxylic acid, both of which render protein denaturing difficult. When a spore’s milieu once again provides favorable conditions (nutrient medium, temperature, osmotic pressure, etc.) it returns to the vegetative state in which spore-forming bacteria can reproduce.

The Physiology of Metabolism and Growth in Bacteria

• Human pathogenic bacteria are chemosynthetic and organotrophic (chemo-organotrophic). They derive energy from the breakdown of organic nutrients and use this chemical energy both for resynthesis and secondary activities. Bacteria oxidize nutrient substrates by means of either respiration or fermentation. In respiration, O2 is the electron and proton acceptor, in fermentation an organic molecule performs this function. Human pathogenic bacteria are classified in terms of their O2 requirements and tolerance as facultative anaerobes, obligate aerobes, obligate anaerobes, or aerotolerant anaerobes. Nutrient broth or agar is used to cultivate bacteria. Nutrient agar contains the inert substrate agarose, which liquefies at 100 8C and gels at 45 8C. Selective and indicator mediums are used frequently in diagnostic bacteriology.

• Bacteria reproduce by means of simple transverse binary fission. The time required for complete cell division is called generation time. The in-vitro generation time of rapidly proliferating species is 15–30 minutes. This time is much longer in vivo. The growth curve for proliferation in nutrient broth is normally characterized by the phases lag, log (or exponential) growth, stationary growth, and death

Bacterial Metabolism

Types of Metabolism

• Metabolism is the totality of chemical reactions occurring in bacterial cells. They can be subdivided into anabolic (synthetic) reactions that consume energy and catabolic reactions that supply energy. In the anabolic, endergonic reactions, the energy requirement is consumed in the form of light or chemical energy—by photosynthetic or chemosynthetic bacteria, respectively. Catabolic reactions supply both energy and the basic structural elements for synthesis of specific bacterial molecules. Bacteria that feed on inorganic nutrients are said to be lithotrophic, those that feed on organic nutrients are organotrophic.

• Human pathogenic bacteria are always chemosynthetic, organotrophic bacteria (or chemo-organotroph

Catabolic Reactions

• Organic nutrient substrates are catabolized in a wide variety of enzymatic processes that can be schematically divided into four phases:

• Digestion. Bacterial exoenzymes split up the nutrient substrates into smaller molecules outside the cell. The exoenzymes represent important pathogenicity factors in some cases.

• Uptake. Nutrients can be taken up by means of passive diffusion or, more frequently, specifically by active transport through the membrane(s). Cytoplasmic membrane permeases play an important role in these Proces.

• Preparation for oxidation. Splitting off of carboxyl and amino groups, phosphorylation, etc.

• Oxidation. This process is defined as the removal of electrons and H+ ions. The substance to which the H2 atoms are transferred is called the hydrogen acceptor. The two basic forms of oxidation are defined by the final hydrogen acceptor.

• Respiration. Here oxygen is the hydrogen acceptor. In anaerobic respiration, the O2 that serves as the hydrogen acceptor is a component of an inorganic salt.

• Fermentation. Here an organic compound serves as the hydrogen acceptor. The main difference between fermentation and respiration is the energy yield, which can be greater from respiration than from fermentation for a given nutrient substrate by as much as a factor of 10. Fermentation processes involving microorganisms are designated by the final product, e.g., alcoholic fermentation, butyric acid fermentation, etc. The energy released by oxidation is stored as chemical energy in the form of a thioester (e.g., acetyl-CoA) or organic phosphates (e.g., ATP).

• The role of oxygen. Oxygen is activated in one of three ways: & Transfer of 4e– to O2, resulting in two oxygen ions (2 O2–). & Transfer of 2e– to O2, resulting in one peroxide anion (1 O2 2–). & Transfer of 1e– to O2, resulting in one superoxide anion (1 O2 –).

• Hydrogen peroxide and the highly reactive superoxide anion are toxic and therefore must undergo further conversion immediately.

• Facultative anaerobes. These bacteria can oxidize nutrient substrates by means of both respiration and fermentation.

• Obligate aerobes. These bacteria can only reproduce in the presence of O2.

• Obligate anaerobes. These bacteria die in the presence of O2. Their metabolism is adapted to a low redox potential and vital enzymes are inhibited by O2.

• Aerotolerant anaerobes. These bacteria oxidize nutrient substrates without using elemental oxygen although, unlike obligate anaerobes, they can tolerate it.

• Basic mechanisms of catabolic metabolism. The principle of the biochemical unity of life asserts that all life on earth is, in essence, the same. Thus, the catabolic intermediary metabolism of bacteria is, for the most part, equivalent to what takes place in eukaryotic cells. The reader is referred to textbooks of general microbiology for exhaustive treatment of the pathways of intermediary bacterial metabolism.

Anabolic Reactions

• It is not possible to go into all of the biosynthetic feats of bacteria here. Suffice it to say that they are, on the whole, quite astounding. Some bacteria (E. coli) are capable of synthesizing all of the complex organic molecules that they are comprised of, from the simplest nutrients in a very short time. These capacities are utilized in the field of microbiological engineering. Antibiotics, amino acids, and vitamins are produced with the help of bacteria. Some bacteria are even capable of using aliphatic hydrocarbon compounds as an energy source. Such bacteria can “feed” on paraffin or even raw petroleum. It is hoped that the metabolic capabilities of these bacteria will help control the effects of oil spills in surface water. Bacteria have also been enlisted in the fight against hunger: certain bacteria and fungi are cultivated on aliphatic hydrocarbon substrates, which supply carbon and energy, then harvested and processed into a protein powder (single cell protein). Culturing of bacteria in nutrient mediums based on methanol is another approach being used to produce biome.

Metabolic Regulation

• Bacteria are highly efficient metabolic regulators, coordinating each individual reaction with other cell activities and with the available nutrients as economically and rationally as possible. One form such control activity takes is regulation of the activities of existing enzymes. Many enzymes are allosteric proteins that can be inhibited or activated by the final products of metabolic pathways. One highly economical type of regulation controls the synthesis of enzymes at the genetic transcription or translation level (see the section on the molecular basis of bacterial genetics (p. 169ff.).

Growth and Culturing of Bacteria

Nutrients

• The term bacterial culture refers to proliferation of bacteria with a suitable nutrient substrate. A nutrient medium (Table 3.2) in which chemoorganotrophs are to be cultivated must have organic energy sources (H2 donors) and H2 acceptors. Other necessities include sources of carbon and nitrogen for synthesis of specific bacterial compounds as well as minerals such as sulfur, phosphorus, calcium, magnesium, and trace elements as enzyme activators. Some bacteria also require “growth factors,” i.e., organic compounds they are unable to synthesize themselves. Depending on the bacterial species involved, the nutrient medium must contain certain amounts of O2 and CO2 and have certain pH and osmotic pressure levels.

Growth and Cell Death

• Bacteria reproduce asexually by means of simple transverse binary fission. Their numbers (n) increase logarithmically (n = 2G). The time required for a reproduction cycle (G) is called the generation time (g) and can vary greatly from species to species. Fast-growing bacteria cultivated in vitro have a gen

• oration time of 15–30 minutes. The same bacteria may take hours to repro

• duce in vivo. Obligate anaerobes grow much more slowly than aerobes; this is true in vitro as well. Tuberculosis bacteria have an in-vitro generation time of 12–24 hours. Of course, the generation time also depends on the nutrient content of the medium.

• The so-called normal growth curve for bacteria is obtained by inoculating a nutrient broth with bacteria the metabolism of which is initially quiescent, counting them at intervals and entering the results in a semilow coordinate system (Fig. 3.16). The lag phase (A) is characterized by an increase in bacterial mass per unit of volume, but no increase in cell count. During this phase, the metabolism of the bacteria adapts to the conditions of the nutrient medium. In the following log (or exponential) phase (C), the cell count increases logarithmically up to about 109/ml. This is followed by growth deceleration and transition to the stationary phase (E) due to exhaustion of the nutrients and the increasing concentration of toxic metabolites. Finally, death phase (F) processes begin. The generation time can only be determined during phase C, either graphically or by determining the cell count (n) at two different times and applying the formula:

The Molecular Basis of Bacterial Genetics

• Bacteria possess two genetic structures: the chromosome and the plasmid. Both of these structures consist of a single circular DNA double helix twisted counterclockwise about its helical axis. Replication of this DNA molecule always starts at a certain point (the origin of replication) and is “semiconservative,” that is, one strand in each of the two resulting double strands is conserved. Most bacterial genes code for proteins (polypeptides). Noncoding interposed sequences (introns), like those seen in eukaryotes, are the exception. Certain bacterial genes have a mosaic structure. The phases oftranscription are promoter recognition, elongation, and termination. Many bacterial mRNAs are polycistronic, meaning they contain the genetic information for several polypeptides. Translation takes place on the 70S ribosomes. Special mRNA codons mark the start and stop of polypeptide synthesis. Many genes that code for functionally related polypeptides are grouped together in chromosome or plasmid segments known as operons. The most important regulatory mechanism is the positive or negative control of transcription initiation. This control function may be exercised by individual localized genes, the genes of an operon or genes in a regulon.

The Structure of Bacterial DNA

• A bacterium’s genetic information is stored in its chromosome and plasmids. Each of these structures is made of a single DNA double helix twisted to the right, then additionally twisted to the left about its helical axis (supercoiled, see p. 148ff. and Fig. 3.17). Plasmids consisting of linear DNA also occur, although this is rare. This DNA topology solves spatial problems and enables such functions as replication, transcription, and recombination. Some genes are composed of a mosaic of Mini cassettes interconnected by conserved DNA sequences between the cassettes.

Chromosome

• The chromosome corresponds to the nucleoid (p. 148ff.). The E. coli chromosome is composed of 4.63 ! 106 base pairs (bp). It codes for 4288 proteins. The gene sequence is colinear with the expressed genetic products. The noncoding interposed sequences (introns) normally seen in eukaryotic genes are very rare. The chromosomes of E. coli and numerous other pathogenic bacteria have now been completely sequenced.

Plasmids

• The plasmids are autonomous DNA molecules of varying size (3 ! 103 to 4.5 ! 105 bp) localized in the cytoplasm. Large plasmids are usually present in one to two copies per cell, whereas small ones may be present in 10, 40, or 100 copies. Plasmids are not essential to a cell’s survival.

Virulence plasmids

• Carry determinants of bacterial virulence, e.g., enterotoxin genes or hemolysin genes. & Resistance plasmids. 

Carry genetic information bearing on resistance to anti-infective agents

• R plasmids may carry several R genes at once (see also Fig. 3.23, p. 176). Plasmids have also been described that carry both virulence and resistance genes.

DNA Replication

• The identical duplication process of DNA is termed semiconservative because the double strand of DNA is opened up during replication, whereupon each strand serves as the matrix for synthesis of a complementary strand. Thus each of the two new double strands “conserves” one old strand. The doubling of each DNA molecule (replicon) begins at a given starting point, the so-called origin of replication. This process continues throughout the entire fission cycle.

Transcription and Translation

Transcription

• Copying of the sense strand of the DNA into mRNA. The continuous genetic nucleotide sequence is transcribed “colinearly” into mRNA. This principle of colinearity applies with very few exceptions. The transcription process can be broken down into the three phases promoter recognition, elongation, and termination. The promoter region is the site where the RNA polymerase begins reading the DNA sequence. A sigma factor is required for binding to the promoter. Sigma factors are proteins that associate temporarily with the RNA polymerase (core enzyme) to form a holoenzyme, then dissociate themselves once the transcription process has begun, making them available to associate once again. Specific sigma factors recognize the standard promoters of most genes. Additional sigma factors, the expression of which depends on the physiological status of the cell, facilitate the transcription of special determinants. Genes that code for functionally related proteins, for example proteins that act together to catalyze a certain metabolic step, are often arranged sequentially at specific locations on the chromosome or plasmid. Such DNA sequences are known as operons (Fig. 3.18). The mRNA synthesized by the transcription of an operon is polycistronic, i.e., it contains the information sequences of several genes. The information sequences are separated by intercistronic regions. Each cistron has its own start and stop codon in the mRNA..

Translation

• Transformation of the nucleotide sequence carried by the mRNA into the polypeptide amino acid sequence at the 70S ribosomes. In principle, bacterial and eukaryotic translation is the same. The enzymes and other factors involved do, however, differ structurally and can therefore be selectively blocked by antibiotics (p. 198ff.).

Regulation of Gene Expression

• Bacteria demonstrate a truly impressive capacity for adapting to their environment. A number of regulatory bacterial mechanisms are known, for example posttranslational regulation, translational regulation, transcription termination, and quorum sensing (see Fig. 1.5, p. 20). The details of all these mechanisms would exceed the scope of this book. The most important is regulation of the initiation of transcription by means of activation or repression, a process not observed in this form in eukaryotes: a single gene, or several genes in an operon at one DNA location, may be affected (see Fig. 3.18). The mechanism that has been investigated most thoroughly is transcriptional regulation of catabolic and anabolic operons by a repressor or activator.

The Genetic Variability of Bacteria

• Changes in bacterial DNA are the result of spontaneous mutations in individual genes as well as recombination processes resulting in new genes or genetic combinations. Based on the molecular mechanisms involved, bacterial recombination's are classified as homologous, site-specific, and transpositional. The latter two in particular reflect the high level of mobility of many genes and have made essential contributions to the evolution of bacteria. Although sexual heredity is unknown in bacteria, they do make use of the mechanisms of intercellular transfer of genomic material known as parasexual processes. Transformation designates transfer of DNA that is essential chemically pure from a donor into a receptor cell. In transduction, bacteriophages serve as the vehicles for DNA transport. Conjugation is the transfer of DNA by means of cell-to-cell contact. This process, made possible by conjugative plasmids and transposons, can be a high-frequency one and may even occur between partners of different species, genera, or families. The transfer primarily involves the conjugative elements themselves. Conjugative structures carrying resistance or virulence genes are of considerable medical significance. The processes of restriction and modification are important factors limiting genetic exchange among different taxa. Restriction is based on the effects of restriction endonucleases capable of specific excision of foreign DNA sequences. These enzymes have become invaluable tools in the field of genetic engineering.

Molecular Mechanisms of Genetic Variability

Spontaneous Mutation

• In the year 1943, Luria and Delbra¨ ck used the so-called fluctuation test to demonstrate that changes in the characteristics of bacterial populations were the results of rare, random mutations in the genes of individual cells, which then were selected. Such mutations may involve substitution of a single nucleotide, frameshifts, deletions, inversions, or insertions. The frequency of mutations is expressed as the mutation rate, which is defined as the probability of mutation per gene per cell division. The rate varies depending on the gene involved and is approximately 10–6 to 10–10. Mutation rates may increase drastically due to mutagenic factors such as radioactivity, UV radiation, alkylating chemicals, etc.

Recombination

• The term recombination designates processes that lead to the restructuring of DNA, formation of new genes or genetic combinations. Homologous (generalized) recombination. A precise exchange of DNA between corresponding sequences. Several enzymes contribute to the complex breakage and reunion process involved, the most important being the Reca enzyme and another the Rebcca nuclease. Fig. 1.2 (p. 14) shows an example of homologous recombination resulting in the exchange of Mini cassettes between two genes.

• Site-specific recombination. Integration or excision of a sequence in or from target DNA. Only a single sequence of a few nucleotides of the integrated DNA needs to be homologous with the recombination site on the target DNA. The integration of bacteriophage genomes is an example of what this process facilitates (p. 184f.) Integration of several determinants of antibiotic resistance in one integrum can also utilize this process (Fig. 3.19). Resistance integrous may be integrated in transposable DNA.

• Transposition. The transposition process does not require the donor and target DNA to be homologous. DNA sequences can either be transposed to a different locus on the same molecule or to a different replicon. Just as in site specific recombination, transposition has always played a major role in the evolution of multi-resistance plasmids.

ntercellular Mechanisms of Genetic Variability

• Although bacteria have no sexual heredity in the strict sense, they do have mechanisms that allow for intercellular DNA transfer. These mechanisms, which involve a unilateral transfer of genetic information from a donor cell to a receptor cell, are subsumed under the term Par asexuality.

Transformation

• Transfer of “naked” DNA. In 1928, Griffith demonstrated that the ability to produce a certain type of capsule could be transferred between different pneumococci. Then Avery showed in 1944 that the transforming principle at work was DNA. This transformation process has been observed mainly in the genera Streptococcus, Neisseria, Helicobacter and Hemophilus.

Transduction

• Transfer of DNA from a donor to a receptor with the help of transport bacteriophages.

• Bacteriophages are viruses that infect bacteria (p.182ff.). During their replication process, DNA sequences from the host bacterial cell may replace all or part of the genome in the phage head. Such phage particles are then defective. They can still dock on receptor cells and inject their DNA, but the infected bacterial cell will then neither produce new phages nor be destroyed.

Conjugation

• Conjugation is the transfer of DNA from a donor to a receptor in a conjugal process involving cell-to-cell contact. Conjugation is made possible by two genetic elements: the conjugative plasmids and the conjugative transposons.

• In the conjugation process, the conjugative elements themselves are what are primarily transferred. However, these elements can also mobilize chromosomal genes or otherwise nontransferable plasmids. Conjugation is seen frequently in Gram-negative rods (Enterobacteriaceae), in which the phenomenon has been most thoroughly researched, and enterococci.

• The F-factor in Escherichia coli. This is the prototype of a conjugative plasmid. This factor contains the so-called tra (transfer) genes responsible both for the formation of conjugal pili on the surface of F cells and for the transfer process. The transfer of the conjugative plasmid takes place as shown here in schematic steps.

• Occasional integration of the F factor into the chromosome gives it the conjugative properties of the F factor. Such an integration produces a sort of giant conjugative element, so that chromosomal genes can also be transferred by the same mechanism. Cells with an integrated F factor are therefore called Hfr (“high frequency of recombination”) cells.

• Conjugative resistance and virulence plasmids. Conjugative plasmids that carry determinants coding for antibiotic resistance and/or virulence in addition to the tar genes and Repa are of considerable medical importance. Three characteristics of conjugative plasmids promote a highly efficient horizontal spread of these determinant factors among different bacteria:

• High frequency of transfer. Due to the “transfer replication” mechanism, each receptor cell that has received a conjugative plasmid automatically becomes a donor cell. Each plasmid-positive cell is also capable of multiple plasmid transfers to receptor cells.

• Wide range of hosts. Many conjugative plasmids can be transferred between different taxonomic species, genera, or even families.

• Multiple determinants. Many conjugative plasmids carry several genes determining the phenotype of the carrier cell. The evolution of a hypothetical conjugative plasmid carrying several resistance determinants is shown schematically in Fig. 3.23.

• Conjugative transposons. These are DNA elements (p. 173) that are usually integrated into the bacterial chromosome. They occur mainly in Gram-positive cocci, but have also been found in Gram-negative bacteria (Bacteroides). Conjugative transposons may carry determinants for antibiotic resistance and thus contribute to horizontal resistance transfer. In the transfer process, the transposon is first excised from the chromosome and circularized. Then a single strand of the double helix is cut and the linearized single strand—analogous to the F factor—is transferred into the receptor cell. Conjugative transposons are also capable of mobilizing nonconjugative plasmi.

Restriction, Modification, and Gene Cloning

• The above descriptions of the mechanisms of genetic variability might make the impression that genes pass freely back and forth among the different bacterial species, rendering the species definitions irrelevant. This is not the case. A number of control mechanisms limit these genetic exchange processes. Among the most important are restriction and modification. ReThe above descriptions of the mechanisms of genetic variability might make the impression that genes pass freely back and forth among the different bacterial species, rendering the species definitions irrelevant. This is not the case. A number of control mechanisms limit these genetic exchange processes. Among the most important are restriction and modification.  

Bacteriophages

• Bacteriophages, or simply phages, are viruses that infect bacteria. They possess a protein shell surrounding the phage genome, which with few exceptions is composed of DNA. A bacteriophage attaches to specific receptors on its host bacteria and injects its genome through the cell wall. This forces the host cells to synthesize more bacteriophages. The host cell lyses at the end of this reproductive phase. So-called temperate bacteriophages lysogenize the host cells, whereby their genomes are integrated into the host cell chromosomes as the so-called prophage. The phage genes are inactive in this stage, although the prophage is duplicated synchronously with host cell proliferation. The transition from prophage status to the lytic cycle is termed spontaneous or artificial induction. Some genomes of temperate phages may carry genes which have the capacity to change the phenotype of the host cell. Integration of such a prophage into the chromosome is known as lysogenic conversion.

Definition

• Bacteriophages are viruses the host cells of which are bacteria. Bacteriophages are therefore obligate cell parasites. They possess only one type of nucleic acid, either DNA or RNA, have no enzymatic systems for energy supply and are unable to synthesize proteins on their own.

Morphology

• Similarly, to the viruses that infect animals, bacteriophages vary widely in appearance. Fig. 3.25a shows a schematic view of a T series coli phage. Research on these phages has been particularly thorough. Fig. 3.25b shows an intact T phage next to a phage that has injected its genome

Composition

• Phages are made up of protein and nucleic acid. The proteins form the head, tail, and other morphological elements, the function of which is to protect the phage genome. This element bears the genetic information, the structural genes for the structural proteins as well as for other proteins (enzymes) required to produce new phage particles. The nucleic acid in most phages is DNA, which occurs as a single DNA double strand in, for example, T series phages. These phages are quite complex and have up to 100 different genes. In spherical and filamentous phages, the genome consists of single-stranded DNA (example: UX174). RNA phages are less common.

• Adsorption. Attachment to cell surface involving specific interactions between a phage protein at the end of the tail and a bacterial receptor.

• Penetration. Injection of the phage genome. Enzymatic penetration of the wall by the tail tube tip and injection of the nucleic acid through the tail tube. & Reproduction. Beginning with synthesis of early proteins (zero to two minutes after injection), e.g., the phage-specific replicase that initiates replication of the phage genome. Then follows transcription of the late genes that code for the structural proteins of the head and tail. The new phage particles are assembled in a maturation process toward the end of the reproduction cycle. & Release. This step usually follows the lysis of the host cell with the help of murein hydrolase coded by a phage gene that destroys the cell wall.

• Lysogenic conversion is when the phage genome lysogenizing a cell bears a gene (or several genes) that codes for bacterial rather than viral processes. Genes localized on phage genomes include the gene for diphtheria toxin, the gene for the pyrogenic toxins of group A streptococci and the cholera toxin gene.

The Principles of Antibiotic Therapy

• Specific antibacterial therapy refers to treatment of infections with anti-infective agents directed against the infecting pathogen. The most important group of anti-infective agents are the antibiotics, which are products of fungi and bacteria (Streptomyces's). Anti-infective agents are categorized as having a broad, narrow, or medium spectrum of action. The efficacy, or effectiveness, of a substance refers to its bactericidal or bacteriostatic effect. Anti-infective agents have many different mechanisms of action. Under the influence of sulfonamides and trimethoprim, bacteria do not synthesize sufficient amounts of tetrahydro folic acid. All beta lactam antibiotics irreversibly block the biosynthesis of murein. Rifamycin inhibits the DNA-dependent RNA polymerase (transcription). Aminoglycosides, tetracyclines, and macrolides block translation. All 4-quinolones damage cellular DNA topology by inhibiting bacterial topoisomerases. Due to their genetic variability, bacteria may develop resistance to specific anti-infective agents. The most important resistance mechanisms are inactivating enzymes, resistant target molecules, reduced influx, increased efflux. Resistant strains (problematic bacteria) occur frequently among hospital flora, mainly Enterobacteriaceae, pseudomonads, staphylococci, and enterococci. Laboratory resistance testing is required for specific antibiotic therapy. Dilutions series tests are quantitative resistance tests used to determine the minimum inhibitory concentration (MIC). The disk test is a semiquantitative test used to classify the test bacteria as resistant or susceptible. In combination therapies it must be remembered that the interactions of two or more antibiotics can give rise to an antagonistic effect. Surgical chemoprophylaxis must be administered as a short-term antimicrobial treatment only.

Definitions

• Specific antibacterial therapy designates treatment of infections with anti-infective agents directed against the infecting pathogen (syn. antibacterial chemotherapeutics, antibiotics). One feature of these pharmaceuticals is “selective toxicity,” that is, they act upon bacteria at very low concentration levels without causing damage to the microorganism. The most important group of anti-infective agents is the antibiotics. These natural substances are produced by fungi or bacteria (usually Streptomyces). The term “antibiotic” is often used in medical contexts to refer to all antibacterial pharmaceuticals, not just to antibiotics in this narrower sense. Fig. 3.28 illustrate the relations between an anti-infective agent, the host organism, and a bacterial pathogen. Table 3.4 lists frequently used anti-infective agents. The most important groups (cephalosporins, penicillin's, 4-quinolones, macrolides, tetracyclines) are in bold print. Fig. 3.29 presents the basic chemical structures of the most important anti-infective agents.

Spectrum of Action

• Each anti-infective agent has a certain spectrum of action, which is a range of bacterial species showing natural sensitivity to the substance. Some anti-infective agents have a narrow spectrum of action (e.g., vancomycin). Most, however, have broad spectra like tetracyclines, which affect all eubacteria.

Efficacy

• The efficacy of an anti-infective agent (syn. kinetics of action) defines the way it affects a bacterial population. Two basic effects are differentiated: bacteriostasis, i.e., reversible inhibition of growth, and irreversible bactericidal activity (Fig. 3.30). Many substances can develop both forms of efficacy depending on their concentration, the type of organism, and the growth phase. Many of these drugs also have a post antibiotic effect (PAE) reflecting the damage inflicted on a bacterial population. After the anti-infective agent is no longer present, the bacterial cells not killed require a recovery phase before they can reproduce again. The PAE may last several hours.

• A bacteriostatic agent alone can never completely eliminate pathogenic bacteria from the body’s tissues. “Healing” results from the combined effects of the anti-infective agent and the specific and nonspecific immune defenses of the host organism. In tissues in which this defense system is inefficient (endocardium), in the middle of a purulent lesion where no functional phagocytes are present, or in immunocompromised patients, bactericidal substances must be required. The clinical value of knowing whether an antibacterial drug is bacteriostatic or bactericidal is readily apparent...

• All of the bacteria from an infection focus cannot be eliminated without support from the body’s immune defense system. A bacterial population always includes several cells with phenotypic resistance that is not genotypically founded. These are the so-called per sisters, which occur in in-vitro cultures at frequencies ranging from 1:106 to 1:108 (Fig. 3.30). The cause of such persistence is usually a specific metabolic property of these bacteria that prevents bactericidal substances from killing them. Following discontinuation of therapy, such per sisters can lead to relapses. Infections with L-forms show a special type of persistence when treated with antibiotics that block murein synthesis (p. 156).

Mechanisms of Action

• Table 3.5 provides a concise summary of the molecular mechanisms of action of the most important groups of anti-infective agents.

Pharmacokinetics

• Pharmacokinetics covers the principles of absorption, distribution, and elimination of pharma cons by the microorganism. The reader is referred to standard textbooks of pharmacology for details. The dosage and dosage interval recommendations for antibacterial therapy take into account the widely differing pharmacokinetic parameters of the different anti-infective agents, among them:
• Absorption rate and specific absorption time & Volume of distribution & Protein binding & Serum (blood) concentration & Tissue concentration & Metabolization & Elimination.

Side Effects

• Treatment with anti-infective agents can cause side effects, resulting either from noncompliance with important therapeutic principles or specific patient reactivity. On the whole, such side effects are of minor significance.

• Toxic effects. These effects arise from direct cell and tissue damage in the macroorganism. Blood concentrations of some substances must therefore be monitored during therapy if there is a risk of cumulation due to inefficient elimination (examples: aminoglycosides, vancomycin).

• Toxic effects. These effects arise from direct cell and tissue damage in the macroorganism. Blood concentrations of some substances must therefore be monitored during therapy if there is a risk of cumulation due to inefficient elimination (examples: aminoglycosides, vancomycin).

• Biological side effects. Example: change in or elimination of normal flora, interfering with its function as a beneficial colonizer (see p. 25).

• Clinical resistance. Resistance of bacteria to the concentration of anti-infective agents maintained at the infection site in the marc.

• Natural resistance. Resistance characteristic of a bacterial species, genus, or family.

• Acquired resistance. Strains of sensitive taxa can acquire resistance by way of changes in their genetic material.

• Biochemical resistance. A biochemically detectable resistance observed in strains of sensitive taxa. The biochemical resistance often corresponds to the clinically relevant resistance. Biochemically resistant strains sometimes show low levels of resistance below the clinically defined boundary separating resistant and sensitive strains. Such strains may be medically suscept.

The Problem of Resistance

Definitions

• Clinical resistance. Resistance of bacteria to the concentration of anti-infective agents maintained at the infection site in the macroorganism. Natural resistance. Resistance characteristic of a bacterial species, genus, or family. Acquired resistance. Strains of sensitive taxa can acquire resistance by way of changes in their genetic material. Biochemical resistance. A biochemically detectable resistance observed in strains of sensitive taxa. The biochemical resistance often corresponds to the clinically relevant resistance. Biochemically resistant strains sometimes show low levels of resistance below the clinically defined boundary separating resistant and sensitive strains. Such strains may be medically Susca.

Incidence, Significance

• Problematic bacteria. Strains with acquired resistance are encountered frequently among Enterobacteriaceae, pseudomonads, staphylococci, and enterococci. Specific infection therapy directed at these pathogens is often fraught with difficulties, which explains the label problematic bacteria. They are responsible for most nosocomial infections (p. 342f.). Usually harmless in otherwise healthy persons, they may cause life-threatening infections in highly susceptible, so-called problematic patients. Problematic bacteria are often characterized by multiple resistance. Resistance to anti-infective agents is observed less frequently in nonhospital bacteria..

• Genetic variability. The basic cause of the high incidence of antibiotic resistance experienced with problematic bacteria is the pronounced genetic variability of these organisms, the mechanisms of which are described in the section “Genetic variability” (p. 171 and p. 174). Most important are the mechanisms of horizontal transfer of resistance determinants responsible for the efficient distribution of resistance markers among these bacteria.

• Selection. The origin and distribution of resistant strains is based to a significant extent on selection of resistance variants. The more often anti-infective substances are administered therapeutically, the greater the number of strains that will develop acquired resistance. Each hospital has a characteristic flora reflecting its prescription practice. A physician must be familiar with the resistance characteristics of this hospital flora so that the right Selection. The origin and distribution of resistant strains is based to a significant extent on selection of resistance variants. The more often anti-infective substances are administered therapeutically, the greater the number of strains that will develop acquired resistance. Each hospital has a characteristic flora reflecting its prescription practice. A physician must be familiar with the resistance characteristics of this hospital flora so that the right.

Resistance Mechanisms

• Inactivating enzymes. Hydrolysis or modification of anti-infective agents. 

• Beta-lactamase. Hydrolyze the beta lactam ring of beta lactam antibiotics (see Fig. 3.29). Over 200 different beta lactamases are known. A course classification system is based on the substrate profile in penicillinases and cephalosporinases. Production of some beta lactamases is induced by beta lactams (see p. 169), others are produced constitutively (unregulated).

• Aminoglycosidases. Modify aminoglycosides by means of phosphorylation and nucleotidylation of free hydroxyl groups (phosphotransferases and nucleotidyl transferases) or acetylation of free amino groups (acetyltransferases).

• Chloramphenicol acetyltransferases. Modification, by acetylation, of chloramphenicol.

Resistant target molecules

• Gene products with a low affinity to anti-infective agents are produced based on mutations in natural genes. Example: DNA gyrase subunit A, resistant to 4-quinolones. & Acquisition of a gene that codes for a target molecule with low affinity to anti-infective agents. The resistance protein assumes the function of the sensitive target molecule. Example: methicillin resistance in staphylococci; acquisition of the penicillin-binding protein 2a, which is resistant to betalactam antibiotics and assumes the function of the naturally sensitive penicillin-binding proteins. & Acquisition of the gene for an enzyme that alters the target structure of an anti-infective agent to render it resistant. Example: 23S rRNA methylases; modification of ribosomal RNA to prevent binding of macrolide antibiotics to the ribosome.

Permeability mechanisms

• Reduced influx. Reduction of transport of anti-infective agents from outside to inside through membranes; rare.

• Increased efflux. Active transport of anti-infective agents from inside to outside by means of efflux pumps in the cytoplasmic membrane, making efflux greater than influx; frequent.

Evolution of Resistance to Anti-Infective Agents

• Resistance to anti-infective agents is genetically determined by resistance genes. Many resistance determinants are not new developments in response to the use of medical antibiotics but developed millions of years ago in bacteria with no human associations. The evolutionary process is therefore a “nonanthropogenic” one. The determinants that code for resistance to anti-infective agents that are not antibiotics did develop after the substances began to be used in therapy, hence this is “anthropogenic” evolution. Factors contributing to the resistance problem have included the molecular mechanisms of genetic variability (mutation, homologous recombination, site-specific integration, transposition) and the mechanisms of intercellular gene transfer in bacteria (transformation, transduction, conjugation).

• To interpret the results, the MICs or inhibition zones are brought into relation with the substance concentrations present at a site of infection at standard dosage levels. This calculation is based on known averages for various pharmacokinetic parameters (serum concentration, half-life) and pharmacodynamic parameters (bactericidal activity or not, post antibiotic effect.

• The minimum bactericidal concentration (MBC) is the smallest concentration of a substance required to kill 99.9% of the cells in an inoculum. The MBC is determined using quantitative subcultures from the macroscopically unclouded tubes or (microplate) wells of an MIC dilution series.

Combination Therapy

• Combination therapy is the term for concurrent administration of two or more anti-infective agents. Some galenic preparations combine two components in a fixed ratio (example: cotrimoxazole). Normally, however, the in dividual substances in a combination therapy are administered separately. Several different objectives can be pursued with combination therapy:

• Broadening of the spectrum of action. In mixed infections with pathogens of varying resistance; in calculated therapy of infections with unknown, or not yet known, pathogenic flora and resistance characteristics. & Delay of resistance development. In therapy of tuberculosis; when using anti-infective agents against which bacteria quickly develop resistance. & Potentiation of efficacy. In severe infections requiring bactericidal activity at the site of infection. Best-known example: penicillin plus gentamicin in treatment of endocarditis caused by enterococci or streptococci.

Chemoprophylaxis

• One of the most controversial antibiotic uses is prophylactic antibiosis. There are no clear-cut solutions here. There are certain situations in which chemoprophylaxis is clearly indicated and others in which it is clearly contraindicated. The matter must be decided on a case-by-case basis by weighing potential benefits against potential harm (side effects, superinfections with highly virulent and resistant pathogens, selection of resistant bacteria).

• Chemoprophylaxis is considered useful in malaria, rheumatic fever, pulmonary cystic fibrosis, recurring pyelonephritis, following intensive contact with meningococci carriers, before surgery involving massive bacterial contamination, in heavily immunocompromised patients, in cardiac surgery or in femoral amputations due to circulatory problems. Chemoprophylaxis aimed at preventing a postsurgical infection should begin a few hours before the operation and never be continued for longer than 24–72 hours.

Immunomodulators

• Despite the generally good efficacy of anti-infective agents, therapeutic success cannot be guaranteed. Complete elimination of bacterial pathogens also requires a functioning immune defense system. In view of the fact that the number of patients with severe immunodeficiencies is on the rise, immunomodulators are used as a supportive adjunct to specific antibiotic therapy in such patients. Many of these “cytokines” (see p. 77ff.) produced by the cells of the immune system can now be produced as “recombinant proteins.” Myelopoietic growth factors have now been successfully used in patients suffering from neutropenia. Additional immunomodulators are also available, e.g., interferon gamma (IFNc) and interleukin 2 (IL-2).

Laboratory Diagnosis

• Infections can be diagnosed either directly by detection of the pathogen or components thereof or indirectly by antibody detection methods. The reliability of laboratory results is characterized by the terms sensitivity and specificity, their value is measured in terms of positive to negative predictive value. These predictive values depend to a great extent on prevalence. In direct laboratory diagnosis, correct material sampling and adequate transport precautions are an absolute necessity. The classic methods of direct laboratory diagnosis include microscopy and culturing. Identification of pathogens is based on morphological, physiological, and chemical characteristics. Among the latter, the importance of detection of pathogen-specific nucleotide sequences is constantly increasing. Development of sensitive test systems has made direct detection of pathogen components in test materials possible in some cases. The molecular biological methods used are applied with or without amplification of the sequence sought as the case warrants. Direct detection can also employ polyclonal or monoclonal antibodies to detect and identify antigens.

Preconditions, General Methods, Evaluation

Preconditions

• The field of medical microbiology dealing with laboratory diagnosis of infectious diseases is known as diagnostic or clinical microbiology. Modern medical practice, and in particular hospital-based practice, is inconceivable without the cooperation of a special microbiological laboratory. To ensure optimum patient benefit, the physician in charge of treatment and the laboratory staff must cooperate closely and efficiently. The preconditions include a basic knowledge of pathophysiology and clinical infectiology on the part of the laboratory staff and familiarity with the laboratory work on the part of the treating physician. The following sections provide a brief rundown on what physicians need to know about laboratory procedures.

General Methods and Evaluation

• An infectious disease can be diagnosed directly by finding the causal pathogen or its components or products. It can also be diagnosed indirectly by means of antibody detection (Chapter 2, p. 121ff.). The accuracy and value of each of the available diagnostic methods are characterized in terms of sensitivity, specificity, and positive or negative predictive value. These parameters are best understood by reference to a 2 ! 2 table (Table 3.6). By inserting fictitious numbers into the 2 ! 2 table, it readily becomes apparent that a positive predictive value will fall rapidly, despite high levels of specificity and sensitivity, if the prevalence level is low (Baye’s theorem).

Sampling and Transport of Test Material

• It is very important that the material to be tested be correctly obtained (sampled) and transported. In general, material from which the pathogen is to be isolated should be sampled as early as possible before chemotherapy is begun. Transport to the laboratory must be carried out in special containers provided by the institutes involved, usually containing transport mediums— either enrichment mediums (e.g., blood culture bottle), selective growth mediums or simple transport mediums without nutrients. An invoice must be attached to the material containing the information required for processing (using the form provided).

Material from the respiratory tract:

• Swab smear from tonsils. — Sinus flushing fluid. — Pulmonary secretion. Expectorated sputum is usually contaminated with saliva and the flora of the oropharynx. Since these contaminations include pathogens that may cause infections of the lower respiratory tract organs, the value of positive findings would be limited. The material can be considered unsuitable for diagnostic testing if more than 25 oral epithelia are present per viewing frame at 100! magnification. Morning sputum from flushing the mouth or after induction will result in suitable samples. Sputum is not analyzed for anaerobes. — Useful alternatives to expectorated sputum include biomicroscopically sampled bronchial secretion, flushing fluid from bronchoalveolar lavage (BAL), transtracheal aspirate or a pulmonary puncture biopsy. These types of material are required if an anaerobe infection is suspected. The material must then be transported in special anaerobe transport containers.

Material from the urogenital tract:

• Urine. Midstream urine is in most cases contaminated with the flora of the anterior urethra, which often corresponds to the pathogen spectrum of urinary tract infections. Bacterial counts must be determined if “contamination” is to be effectively differentiated from “infection.” At counts in morning urine of > –105/ml an infection is highly probable, at counts of < –103 rather improbable. At counts of around 104/ml the test should be repeated. Lower counts may also be diagnostically significant in erythrocytosis. The dipstick method, which can be used in any medical practice, is a simple way of estimating the bacterial count: a stick coated with nutrient medium is immersed in the midstream urine, then incubated. The colony count is then estimated by comparing the result with standardized images.

Blood:

• For a blood culture, at least 10–20 ml of venous blood should be drawn sterilely into one aerobic and one anaerobic blood culture bottle. Sample three times a day at intervals of several hours (minimum interval one hour). — For serology, (2–)5 ml of native blood will usually suffice. Take the initial sample as early as possible and a second one 1–3 weeks later to register any change in the antibody titer.

Pus and wound secretions:

• For surface wounds sample material with smear swabs and transport in preservative transport mediums. Such material is only analyzed for aerobic bacteria. — For deep and closed wounds, liquid material (e.g., pus) should be sampled, if possible, with a syringe. Use special transport mediums for anaerobes.

Material from the gastrointestinal tract:

• Use a small spatula to place a portion of stool about the size of a cherry in liquid transport medium for shipment. — Transport duodenal juice and bile in sterile tubes. Use special containers if anaerobes are suspected.

Cerebrospinal fluid, puncture biopsies, exudates, transudates:

• Ensure sampling sterility. Use special containers if anaerobes are suspected.

Microscopy

• Bacteria are so small that a magnification of 1000! is required to view them properly, which is at the limit of light microscope capability. At this magnification, bacteria can only be discerned in a preparation in which their density is at least 104–105 bacteria per ml.

• Native preparations, with or without vital staining, are used to observe living bacteria. The poor contrast of such preparations makes it necessary to amplify this aspect (dark field and phase contrast microscopy). Native preparations include the coverslip and suspended drop types.

• Stained preparations are richer in contrast so that bacteria are readily recognized in an illuminated field at 1000! The staining procedure kills the bacteria. The material is first applied to a slide in a thin layer, dried in the air, and fixed with heat or methyl alcohol. Simple and differential staining techniques are used. The best-known simple staining technique employs methylene blue. Gram staining is the most important differential technique (Table 3.7): Gram-positive bacteria stain blue-violet, Gram-negative bacteria stain red. The Gram-positive cell wall prevents alcohol elution of the stain iodine complex. In old cultures in which autolytic enzymes have begun to break down the cell walls, Gram-positive cells may test Gram-negative (“Gram-labile” bacteria).

Culturing Methods

• Types of nutrient mediums. Culturing is required in most cases to detect and identify bacteria. Almost all human pathogen bacteria can be cultivated on nutrient mediums. Nutrient mediums are either liquid (nutrient broth) or gelatinous (nutrient agar, containing 1.5–2% of the polysaccharide agarose). Enrichment mediums are complex mediums that encourage the proliferation of many different bacterial species. The most frequently used enrichment medium is the blood agar plate containing 5% whole blood. Selective mediums allow only certain bacteria to grow and suppress the reproduction of others. Indicator mediums are used to register metabolic processes.

• Proliferation forms. Most bacteria show diffuse proliferation in liquid mediums. Some proliferate in “crumbs,” other form a grainy bottom sediment, yet others a biofilm skin at the surface (pseudomonads). Isolated colonies are observed to form on, or in, nutrient agar if the cell density is not too high. These are pure cultures, since each colony arises from a single bacterium or colony-forming unit (CFU). The pure culture technique is the basis of bacteriological culturing methods. The procedure most frequently used to obtain isolated colonies is fractionated inoculation of a nutrient agar plate.

• Conditions required for growth. The optimum proliferation temperature for most human pathogen bacteria is 37 8C. Bacteria are generally cultured under atmospheric conditions. It often proves necessary to incubate the cultures in 5% CO2. Obligate anaerobes must be cultured in a milieu with a low redox potential. This can be achieved by adding suitable reduction agents to the nutrient broth or by proliferating the cultures under a gas atmosphere from which most of the oxygen has been removed by physical, chemical, or biological means.

Identification of Bacteria

• The essential principle of bacterial identification is to assign an unknown culture to its place within the taxonomic classification system based on as few characteristics as possible and as many as necessary (Table 3.8).

• Morphological characteristics, including staining, are determined under the microscope.

• Physiological characteristics are determined with indicator mediums. Commercially available miniaturized systems are now frequently used for this purpose (Fig. 3.36).

Morphological characteristics

• Form (sphere, rod, spiral) Size; pseudogroupings (clusters, chains, diplococci) Staining (Gram-positive, Gram-negative); flagella (presence, arrangement); capsule (yes, no); spores (form, within cell formation).

Physiological characteristics

• Respiratory chain enzymes (oxidases, catalases) Enzymes that break down carbohydrates, alcohols, glycosides (e.g., betagalactosidase) Protein metabolism enzymes (e.g., gelatinase, collagenase) Amino acid metabolism enzymes (e.g., decarboxylases, deaminases, urease) Other enzymes: hemolysins, lipases, lecithinases, DNases, etc. End products of metabolism (e.g., organic acids detected by gas chromatography) Resistance/sensitivity to chemical noxae Characteristics of anabolic metabolism (e.g., citrate as sole source of C).

Chemical characteristics

• DNA structure (base sequences) Structure of cell wall murein Antigen structure: fine structures detectable with antibodies (e.g., flagellar protein or polysaccharides of the cell wall or capsule) Fatty acids in membranes and cell wall; analysis using different chromatographic methods.

• Chemical characteristics have long been in use to identify bacteria, e.g., in detection of antigen structures. Molecular genetic methods (see below) will play an increasing role in the future.

Molecular Methods

• The main objective of the molecular methods of bacterial identification is direct recognition of pathogen-specific nucleotide sequences in the test material. These methods are used in particular in the search for bacteria that are not culturable, are very difficult to culture, or proliferate very slowly. Of course, they can also be used to identify pure bacterial cultures (see above). In principle, any species-specific sequence can be used for identification, but the specific regions of genes coding for 16S rRNA and 23S rRNA are particularly useful in this respect. The following methods are used:

• DNA probes. Since DNA is made up of two complementary strands of nucleic acids, it is possible to detect single-strand sequences with the hybridization technique using complementary marking of single strands. The probes can be marked with radioactivity ( 32P, 35S) or nonradioactive reporter molecules (biotin, digoxygenin):

• Solid phase hybridization. The reporter molecule or probe is fixed to a nylon or nitrocellulose membrane (colony blot technique, dot blot technique). — Liquid phase hybridization. The reporter molecule and probe are in a solute state. — In-situ hybridization. Detection of bacterial DNA in infected tissue.

• Amplification. The main objective here is to increase the sensitivity level so as to find the “needle in a haystack.” A number of techniques have been developed to date, which can be classified in three groups:

• Amplification of the target sequence. The oldest and most important among the techniques in this group is the polymerase chain reaction (PCR), which is described on p. 409f.). With “real time PCR,” a variant of PCR, the analysis can be completed in 10 minutes. — Probe amplification. — Signal amplification.

Direct Detection of Bacterial Antigens

• Antigens specific for particular species or genera can be detected directly by means of polyclonal or (better yet) monoclonal antibodies present in the test material. This allows for rapid diagnosis. Examples include the detection of bacterial antigens in cerebrospinal fluid in cases of acute purulent meningitis, detection of gonococcal antigens in secretion from the urogenital tract, and detection of group A streptococcal antigen in throat smear material. These direct methods are not, however, as sensitive as the classic culturing methods. Absorbances, agglutination, and latex agglutination tests are frequently used in direct detection. In the agglutination methods, the antibodies with the Fc components are fixed either to killed staphylococcal protein A or to latex particles.

Diagnostic Animal Tests

• Animal testing is practically a thing of the past in diagnostic bacteriology. Until a few years ago, bacterial toxins (e.g., diphtheria toxin, tetanus toxin, botulinus toxin) were confirmed in animal tests. Today, molecular genetic methods are used to detect the presence of the toxin gene, which process usually involves an amplification step.

Bacteriological Laboratory Safety

• Microbiologists doing diagnostic work will of course have to handle potentially pathogenic microorganisms and must observe stringent regulations to avoid risks to themselves and others. Laboratory safety begins with suitable room designs and equipment (negative-pressure lab rooms, safety hoods) Microbiologists doing diagnostic work will of course have to handle potentially pathogenic microorganisms and must observe stringent regulations to avoid risks to themselves and others. Laboratory safety begins with suitable room designs and equipment (negative-pressure lab rooms, safety hoods).

Taxonomy and Overview of Human Pathogenic Bacteria

• Taxonomy includes the two disciplines of classification and nomenclature. The bacteria are classified in a hierarchic system based on phenotypic characteristics (morphological, physiological, and chemical characteristics). The basic unit is the species. Similar and related species are classified in a single genus and related genera are placed in a single family. Classification in yet higher taxa often takes practical considerations into account, e.g., division into “descriptive sections.” A species is designated by two Latin names, the first of which denotes the genus, both together characterizing the species. Family names end in -aceae. Table 3.9 provides an overview of human pathogenic bacteria.

Classification

• Bacteria are grouped in the domain bacteria to separate them from the domains archaea and eucarya (see p. 5). Within their domain, bacteria are further broken down into taxonomic groups (taxa) based on relationships best elucidated by knowledge of the evolutionary facts. However, little is known about the phylogenetic relationships of bacteria, so their classification is often based on similarities among phenotypic characteristics (phenetic relationships). These characteristics are morphological, physiological (metabolic), or chemical (see Table 3.8, p. 215) in nature. The role of chemical characteristics in classification is growing in importance, for instance, murein composition or the presence of certain fatty acids in the cell wall. DNA and RNA structure is highly important in classification. DNA composition can be roughly estimated by determining the proportions of the bases: mol/l of guanine + cytosine (GC). The GC content (in mol%) of human pathogenic bacteria ranges from 25% to 70%. Measurement of how much heterologous duplex DNA is formed, or of RNA-DNA hybrids, provides information on the similarity of different bacteria and thus about their degree of relationship. Another highly useful factor in determining phylogenetic relationship is the sequence analysis of the (16S/23S) rRNA or (16S/23S) rDNA. This genetic material contains highly conserved sequences found in all bacteria alongside sequences characteristic of the different taxa.

Nomenclature

• The rules of bacterial nomenclature are set out in the International Code for the Nomenclature of Bacteria. A species is designated with two latinized names, the first of which characterizes the genus and the second the species. Family names always end in -aceae. Taxonomic names approved by the “International Committee of Systematic Bacteriology” are considered official and binding. In medical practice, short handles have become popular in many cases, for instance gonococci instead of Neisseria gonorrhea or pneumococci (or even “strep pneumos”) instead of Streptococcus pneumoniae.