Chapter -1
General Aspects of Medical Microbiology
The History of Infectious Diseases
Infectious diseases have been known for thousands of years, although accurate information on their etiology has only been available for about a century. In the medical teachings of Hippocrates, the cause of infections occurring frequently in a certain locality or during a certain period (epidemics) was sought in “changes” in the air according to the theory of miasmas. This concept, still reflected in terms such as “swamp fever” or “malaria,” was the predominant academic opinion until the end of the 19th century, despite the fact that the Dutch cloth merchant A. van Leeuwenhoek had seen and described bacteria as early as the 17th century, using a microscope he built himself with a single convex lens and a very short focal length. At the time, general acceptance of the notion of “spontaneous generation”—creation of life from dead organic material—stood in the way of implicating the bacteria found in the corpses of infection victims as the cause of the deadly diseases. It was not until Pasteur disproved the doctrine of spontaneous generation in the second half of the 19th century that a new way of thinking became possible. By the end of that century, microorganisms had been identified as the causal agents in many familiar diseases by applying the Henle-Koch postulates formulated by R. Koch in 1890.
The Henle–Koch Postulates
The postulates can be freely formulated as follows:
The microorganism must be found under conditions corresponding to the pathological changes and clinical course of the disease in question.
It must be possible to cause an identical (human) or similar (animal) disease with pure cultures of the pathogen.
The pathogen must not occur within the framework of other diseases as an “accidental parasite.”
These postulates are still used today to confirm the cause of an infectious disease. However, the fact that these conditions are not met does not necessarily exclude a contribution to disease etiology by a pathogen found in context. In particular, many infections caused by subcellular entities do not fulfill the postulates in their classic form.
The Present
The frequency and deadliness of infectious diseases throughout thousands of years of human history have kept them at the focus of medical science. The development of effective preventive and therapeutic measures in recent decades has diminished, and sometimes eliminated entirely, the grim epidemics of smallpox, plague, spotted fever, diphtheria, and other such contagions. Today we have specific drug treatments for many infectious diseases. As a result of these developments, the attention of medical researchers was diverted to other fields: it seemed we had tamed the infectious diseases. Recent years have proved this assumption false. Previously unknown pathogens causing new diseases are being found and familiar organisms have demonstrated an ability to evolve new forms and reassert themselves. The origins of this reversal are many and complex: human behavior has changed, particularly in terms of mobility and nutrition. Further contributory factors were the introduction of invasive and aggressive medical therapies, neglect of established methods of infection control and, of course, the ability of pathogens to make full use of their specific genetic variability to adapt to changing conditions. The upshot is that physicians in particular, as well as other medical professionals and staff, urgently require a basic knowledge of the pathogens involved and the genesis of infectious diseases if they are to respond effectively to this dynamism in the field of infectiology. The aim of this textbook is to impart these essentials to them.
Pathogens
Subcellular Infectious Entities
& Prions (proteinaceous infectious particles). The evidence indicates that prions are protein molecules that cause degenerative central nervous system (CNS) diseases such as Creutzfeldt-Jakob disease, kuru, scrapie in sheep, and bovine spongiform encephalopathy (BSE) (general term: transmissible spongiform encephalopathies [TSE]).
Viruses. Ultramicroscopic, obligate intracellular parasites that:
contain only one type of nucleic acid, either DNA or RNA,
possess no enzymatic energy-producing system and no protein-synthesizing apparatus, and
— force infected host cells to synthesize virus particles
Prokaryotic and Eukaryotic Microorganisms
According to a proposal by Woese that has been gaining general acceptance in recent years, the world of living things is classified in the three domains bacteria, archaea, and eugarie. In this system, each domain is subdivided into
Bacteria, Archaea, Eucarya
Archaea. This domain includes forms that live under extreme environmental conditions, including thermophilic, hyperthermophiles, halophilic, and methanogenic microorganisms. The earlier term for the archaea was archaebacteria (ancient bacteria), and they are indeed a kind of living fossil. Thermophilic archaea thrive mainly in warm, moist biotopes such as the hot springs at the top of geothermal vents. The hyperthermophiles archaea, a more recent discovery, live near deep-sea volcanic plumes at temperatures exceeding 100 8C.
Eucarya. This domain includes all life forms with cells possessing a genuine nucleus. The plant and animal kingdoms (animals and plan tales) are all eukaryotic life forms. Pathogenic eukaryotic microorganisms include fungal and protozoan species.
Bacteria
Chlamydia. These organisms are obligate intracellular parasites that are able to reproduce in certain human cells only and are found in two stages: the infectious, nonreproductive particles called elementary bodies (0.3 lm) and the noninfectious, intracytoplasmic, reproductive forms known as initial (or reticulate) bodies (1 lm).
Rickettsia. These organisms are obligate intracellular parasites, rod-shaped to coccoid, that reproduce by binary transverse fission. The diameter of the individual cell is from 0.3–1 lm.
Mycoplasmas. Mycoplasmas are bacteria without rigid cell walls. They are found in a wide variety of forms, the most common being the coccoid cell (0.3–0.8 lm). Threadlike forms also occur in various lengths. Fungi and Protozoa & Fungi. Fungi (Lycophyte) are nonmotile eukaryotes with rigid cell walls and a classic cell nucleus. They contain no photosynthetic pigments and are carbon heterotrophic, that is, they utilize various organic nutrient substrates (in contrast to carbon autotrophic plants). Of more than 50 000 fungal species, only about 300 are known to be human pathogens. Most fungal infections occur as a result of weakened host immune defenses.
Protozoa. Protozoa are microorganisms in various sizes and forms that may be free-living or parasitic. They possess a nucleus containing chromosomes and organelles such as mitochondria (lacking in some cases), a idioplasmic reticulum, pseudopods, flagella, cilia, kinetoplasts, etc. Many parasitic protozoa are transmitted by arthropods, whereby multiplication and transformation into the infectious stage take place in the vector.
Animals
Helminths. Parasitic worms belong to the animal kingdom. These are metazoan organisms with highly differentiated structures. Medically significant groups include the trematodes (flukes or flatworms), cestodes (tapeworms), and nematodes (roundworms).
Arthropods. These animals are characterized by an external chitin skeleton, segmented bodies, jointed legs, special mouthparts, and other specific features. Their role as direct causative agents of diseases is a minor one (mites, for instance, cause scabies) as compared to their role as vectors transmitting viruses, bacteria, protozoa, and helminth
Host–Pathogen Interactions
The factors determining the genesis, clinical picture and outcome of an infection include complex relationships between the host and invading organisms that differ widely depending on the pathogen involved. Despite this variability, a number of general principles apply to the interactions between the invading pathogen with its aggression factors and the host with its defenses. Since the pathogenesis of bacterial infectious diseases has been researched very thoroughly, the following summary is based on the host–invader interactions seen in this type of infection.
The determinants of bacterial pathogenicity and virulence can be outlined as follows:
Adhesion to host cells (adhesins).
Breaching of host anatomical barriers (invasions') and colonization of tissues (aggression's).
Strategies to overcome nonspecific defenses, especially antiphagocytic mechanisms (impedes).
Strategies to overcome specific immunity, the most important of which is production of IgA proteases (impedes), molecular mimicry, and immunogen variability
Damage to host tissues due to direct bacterial cytotoxicity, exotoxins, and exoenzymes (aggressing).
Damage due to inflammatory reactions in the microorganism: activation of complement and phagocytosis; induction of cytokine production (moulins).
The above bacterial pathogenicity factors are confronted by the following host defense mechanisms:
Nonspecific defenses including mechanical, humoral, and cellular systems. Phagocytosis is the most important process in this context
Specific immune responses based on antibodies and specific reactions of T lymphocytes (see chapter on immunology).
The response of these defenses to infection thus involves the correlation of a number of different mechanisms. Defective defenses make it easier for an infection to take hold. Primary, innate defects are rare, whereas acquired, secondary immune defects occur frequently, paving the way for infections by microorganisms known as “facultative pathogens” (opportunists).
Basic Terminology of Infectiology
The terms pathogenicity and virulence are not clearly defined in their relevance to microorganisms. They are sometimes even used synonymously. It has been proposed that pathogenicity be used to characterize a particular species and that virulence be used to describe the sum of the disease-causing properties of a population (strain) of a pathogenic species
Pathogenicity and virulence in the microorganism correspond to susceptibility in a host species and disposition in a specific host organism, whereby an individual may be anywhere from highly disposed to resistant.
Determinants of Bacterial Pathogenicity and Virulence
There are five groups of potential bacterial contributors to the pathogenesis of infectious diseases:
1. Adhesins. They facilitate adhesion to specific target cells
2. Invasion's. They are responsible for active invasion of the cells of the microorganism
3. Impedes. These components disable host immune defenses in some cases.
4. Aggressions. These substances include toxins and tissue-damaging enzymes.
5. Modulink. Substances that induce excess cytokine production (i.e., lipopolysaccharides of Gram-negative bacteria, superantigens, murein fragments).
Adhesion
When pathogenic bacteria come into contact with intact human surface tissues (e.g., mucosa), they contrive to adhere to receptors on the surface of the target cells by means of various surface structures of their own (attachment pili, attachment fimbriae, adhesion proteins in the outer membrane of Gram-negative bacteria, cell wall-associated proteins in Gram-positive bacteria). This is a specific process, meaning that the adhesion structure (or ligand) and the receptor must fit together like a key in a keyhole.
Invasion and Spread
Invasion. Bacteria may invade a host passively through microtraumas or Macro traumata in the skin or mucosa. On the other hand, bacteria that invade through intact mucosa first adhere to this anatomical barrier, then actively breach it. Different bacterial species deploy a variety of mechanisms to reach this end:
Production of tissue-damaging exoenzymes that destroy anatomical barriers
Parasite-directed endocytosis, initiated by invasions on the surface of the bacterial cells, causes the cytoskeleton of the epithelial cell to form pseudopods that bring about endocytose
Phagocytosis of enteropathogenic bacteria by M cells in the intestinal mucosa (cells that can ingest substances from the intestinal lumen by way of phagocytosis)
Spread.
Local tissue spread beginning at the portal of entry, helped along by tissue-damaging exoenzymes (hyaluronidase, collagenase, elastase, and other proteases).
Cell-to-cell spread. Bacteria translocated into the intracellular space by endocytosis cause actin to condense into filaments, which then array at one end of the bacterium and push up against the inner side of the cell membrane. This is followed by fusion with the membrane of the neighboring tissue cell, whereupon the bacterium enters the new cell (typical of Listeria and Shigella).
Translocation of macrophage-resistant bacteria with macrophages into intestinal lymphoid tissue following their ingestion by M cells.
Lymphopenias or hematogenous generalization. The bacteria then invade organs for which they possess a specific tropism.
Strategies against Nonspecific Immunity
Establishment of a bacterial infection in a host presupposes the capacity of the invaders to overcome the host’s nonspecific immune defenses. The most important mechanisms used by pathogenic bacteria are:
Anti phagocytosis
Capsule. Renders phagocytosis more difficult. Capsule components may block alternative activation of complement so that C3b is lacking (ligand for C3b receptor of phagocytes) on the surface of encapsulated bacteria. Microorganisms that use this strategy include Streptococcus pneumoniae and Haemophile's influenzae.
Phagocyte toxins. Examples: leucocidin from staphylococci, streptolysin from streptococci.
Macrophages may be disabled by the type III secretion system (see p. 17) of certain Gram-negative bacteria (for example salmonellae, shigella, Yersinia, and coli bacteria). This system is used to inject toxic proteins into the macrophages.
Inhibition of phagosome-lysosome fusion. Examples: tuberculosis bacteria, gonococci, Chlamydia psittacine.
Inhibition of the phagocytic “oxidative burst.” No formation of reactive O2 radicals in phagocytes. Examples: Legionella pneumophilia, Salmonella typhi.
Serum resistance. Resistance of Gram-negative bacteria to complement. A lipopolysaccharide in the outer membrane is modified in such a way that it cannot initiate alternative activation of the complement system. As a result, the membrane attack complex (C5b6789), which would otherwise lyse holes in the outer membrane, is no longer produced
Siderophores. Siderophores (e.g., enterochelin, aerobactin) are low-molecular-weight iron-binding molecules that transport Fe3+ actively into the intracellular space. They complex with iron, thereby stealing this element from proteins containing iron (transferrin, lactoferrin). The intricate iron transport system is localized in the cytoplasmic membrane, and in Gram negative bacteria in the outer membrane as well. To thrive, bacteria require 10–5 mol/l free iron ions. The free availability of only about 10–20 mol/l iron in human body fluids thus presents a challenge to them.
Strategies against Specific Immunity
Immunotolerance
Molecular mimicry. Molecular mimicry refers to the presence of molecules on the surface of bacteria that are not recognized as foreign by the immune system. Examples of this strategy are the hyaluronic acid capsule of Streptococcus pyogenes or the neuraminic acid capsule of Escherichia coli K1 and serotype B Neisseria meningitidis.
Antigen variation. Some bacteria are characterized by a pronounced variability of their immunogens (= immune antigens) due to the genetic variability of the structural genes coding the antigen proteins. This results in production of a series of antigen variants in the course of an infection that no longer “match” with the antibodies to the “old” antigen. Examples: gonococci can modify the primary structure of the pilin of their attachment pili at a high rate (Fig. 1.2). The borelian that cause relapsing fevers have the capacity to change the structure of one of the adhesion proteins in their outer membrane (vmp = variable major protein), resulting in the typical “recurrences” of fever. Similarly, meningococci can change the chemistry of their capsule polysaccharides (“capsule switching”).
Clinical Disease
The clinical symptoms of a bacterial infection arise from the effects of damaging nixie produced by the bacteria as well as from excessive host immune responses, both nonspecific and specific. Immune reactions can thus potentially damage the host’s health as well as protect it (see Immunology, p.103ff
Cytopathic effect. Obligate intracellular parasites (rickettsia, chlamydia) may kill the invaded host cells when they reproduce
Exotoxins. Pathogenic bacteria can produce a variety of toxins that are either the only pathogenic factor (e.g., in diphtheria, cholera, and tetanus) or at least a major factor in the unfolding of the disease. One aspect the classification and nomenclature of these toxins must reflect is the type of cell affected: cytotoxins produce toxic effects in many different host cells; neurotoxins affect the neurons; enterotoxins affect enterocytes. The structures and mechanisms of action of the toxins are also considered in their classification
AB toxins. They consist of a binding subunit “B” responsible for binding to specific surface receptors on target host cells, and a catalytic subunit “A” representing the active agent. Only cells presenting the “B” receptors are damaged by these toxins.
Membrane toxins. These toxins disrupt biological membranes, either by attaching to them and assembling to form pores, or in the form of phospholipases that destroy membrane structure enzymatically.
Superantigens (see p. 72). These antigens stimulate T lymphocytes and macrophages to produce excessive amounts of harmful cytokines.
Hydrolytic exoenzymes. Proteases (e.g., collagenase, elastase, nonspecific proteases), hyaluronidase, neuraminidase (synonymous with sialidase), lecithin Ase and DNases contribute at varying levels to the pathogenesis of an infection.
Secretion of virulence proteins. Proteins are synthesized at the ribosomes in the bacterial cytoplasm. They must then be secreted through the cytoplasmic membrane, and in Gram-negative bacteria through the outer membrane as well. The secretion process is implemented by complex protein secretion systems (I-IV) with differing compositions and functional pathways. The type III (virulence-related) secretion system in certain Gram-negative bacteria (Salmonella, Shigella, Yersinia, Bordetella, Escherichia coli, Chlamydia) is particularly important in this connection
Cell wall. The endotoxin of Gram-negative bacteria (lipopolysaccharide) plays an important role in the manifestation of clinical symptoms. On the one hand, it can activate complement by the alternative pathway and, by releasing the chemotactic components C3a and C5a, initiate an inflammatory reaction at the infection site. On the other hand, it also stimulates macrophages to produce endogenous pyrogens (interleukin 1, tumor necrosis factor), thus inducing fever centrally. Production of these and other cytokines is increased, resulting in hypotension, intravital coagulation, thrombocyte aggregation and stimulation of granulopoiesis. Increased production of cytokines by macrophages is also induced by soluble murein fragments and, in the case of Gram-positive bacteria, by teichoic acids.
Inflammation. Inflammation results from the combined effects of the nonspecific and specific immune responses of the host organism. Activation of complement by way of both the classic and alternative pathways induces phagocyte migration to the infection site. Purulent tissue necrosis follows. The development of typical granulomas and caseous necrosis in the course of tuberculosis are the results of excessive reaction by the cellular immune system to the immunogens of tuberculosis bacteria. Textbooks of general pathology should be consulted for detailed descriptions of these inflammatory processes
Regulation of Bacterial Virulence
Many pathogenic bacteria are capable of living either outside or inside a host and of attacking a variety of host species. Proliferation in these differing environments demands an efficient regulation of virulence, the aim being to have virulence factors available as required. Four different regulatory mechanisms have been described
DNA changes. The nucleotide sequences of virulence determinants are changed. Examples of this include pilin gene variability involving intracellular recombination as described above in gonococci and inverting a leader sequence to switch genes on and off in the phase variations of H antigens in salmonellae (see p. 284).
Transcriptional regulation. The principle of transcriptional control of virulence determinants is essentially the same as that applying to the regulation of metabolic genes, namely repression and activation (see p. 169f.): — Simple regulation. Regulation of the diphtheria toxin gene has been thoroughly researched. A specific concentration of iron in the cytoplasm activates the diphtheria toxin regulator (Datar). The resulting active repressor prevents transcription of the toxin gene by binding to the promoted region. Other virulence genes can also be activated by regulators using this mechanism.
Complex regulation, virulence regulon. In many cases, several virulence genes are switched on and off by the same regulator protein. The virulence determinants involved are either components of the same operon or are located at different genome sites. Several vir (virulence) genes with promoter regions that respond to the same regulator protein form a so-called Vir regulon. Regulation of the virulence regulon of Bordetella pertussis by means of gene activation is a case in point that has been studied in great detail. This particular regulon comprises over 20 virulence determinants, all controlled by the same vir regulator protein (or began coding region)
Posttranscriptional regulation. This term refers to regulation by mRNA or a posttranslational protein modification.
Quorum sensing. This term refers to determination of gene expression by bacterial cell density (Fig. 1.5). Quorum sensing is observed in both
Gram-positive and Gram-negative bacteria. It denotes a mode of communication between bacterial cells that enables a bacterial population to react analogously to a multicellular organism.
Accumulation of a given density of a low-molecular-weight pheromone (autoinducer) enables a bacterial population to sense when the critical cell density (quorum) has been reached that will enable it to invade the host successfully, at which point transcription of virulence determinants is initiated.
The Genetics of Bacterial Pathogenicity
The virulence genes of pathogenic bacteria are frequently components of mobile genetic elements such as plasmids, bacteriophage genomes, or conjugative transposons (see p. 170ff.). This makes lateral transfer of these genes between bacterial cells possible. Regions showing a high frequency of virulence genes in a bacterial chromosome are called pathogenicity islands (P PIs are found in both Gram-positive and Gram-negative bacteria. These are DNA regions up to 200 kb that often bear several different vir genes and have specific sequences located at their ends (e.g., IS elements) that facilitate lateral translocation of the islands between bacterial cells. The role played by lateral transfer of these islands in the evolutionary process is further underlined by the fact that the GC contents in PIs often differ from those in chromosomal DNA.
Defenses against Infection
A microorganism manifests defensive reactions against invasion by microorganisms in two forms: as specific, acquired immunity and as nonspecific, innate resistance (see also Chapter 2, Basic Principles of Immunology, p. 43).
Nonspecific Defense Mechanisms
Primary defenses. The main factors in the first line of defense against infection are mechanical, accompanied by some humoral and cellular factors. These defenses represent an attempt on the part of the host organism to prevent microorganisms from colonizing its skin and mucosa and thus stave off a generalized invasion.
Secondary defenses. The second line of defense consists of humoral and cellular factors in the blood and tissues, the most important of which are the professional phagocytes.
Phagocytosis. “Professional” phagocytosis is realized by polymorphonuclear, neutrophilic, eosinophilic granulocytes—also known as microphages— and by mononuclear phagocytes (macrophages). The latter also play an important role in antigen presentation (see p. 62). The total microphage cell count in an adult is approximately 2.5! 1012. Only 5% of these cells are located in the blood. They are characterized by a half-life of only a few hours. Microphages contain both primary granules, which are lysosomes containing lysosomal enzymes and cationic peptides, and secondary granules. Both microphages and macrophages are capable of ameboid motility and chemotactic migration, i.e., directed movement along a concentration gradient toward a source of chemotactic substances, in most cases the complement components C3a and C5a. Other potentially chemotactic substances include secretory products of lymphocytes, products of infected and damaged cells or the N-formyl peptides (femto-Phe and femto-Leu-Phe)
a Mechanical factors
Anatomical structure of skin and mucosa
Mucus secretion and mucus flow from mucosa
Muc ciliary movement of the ciliated epithelium in the lower respiratory tract
Digestive tract peristalsis
Urine flow in the urogenital tract
b Humoral factors
Microbicidal effect of the dermal acidic mantle, lactic acid from sweat glands, hydrochloric acid in the stomach, and the unsaturated fatty acids secreted by the sebaceous glands
Complement (alternative activation pathway)
Serum proteins known as acute phase reactants, for example C-reactive protein, haptoglobin, serum amyloid A, fibrinogen, and transferrin (iron-binding protein)
Fibronectin (a nonspecific opsonin); antiviral interferon
Mannose-binding protein: binds to mannose on the outer bacterial surface, thus altering the configuration and triggering alternative activation of complement
c Cellular factors
Normal flora of skin and mucosa
Natural killer cells (large, granulated lymphocytes; null cells)
Professional phagocytes: microphages (neutrophilic and eosinophilic granulocytes); mononuclear phagocytes (macrophages, monocytes
Phagocytes are capable of ingestion of both particulate matter (phagocytosis) and solute matter (pinocytosis). Receptors on the phagocyte membrane initiate contact (Fig. 1.6). Particles adhering to the membrane are engulfed, ingested and deposited in a membrane-bound vacuole, the so-called phagosome, which then fuses with lysosomes to form the phagolysosome. The bacteria are killed by a combination of lysosomal factors:
Mechanisms that require no oxygen. Low pH; acid hydrolases, lysozyme; proteases; defensins (small cationic peptides).
— Mechanisms that require oxygen. Halogenation of essential bacterial components by the myeloperoxidase-H2O2–halide system; production of highly reactive O2 radicals (oxidative burst) such as superoxide anion (O2 –), hydroxyl radical (!OH), and singlet oxygen (1O2).
Specific Defense Mechanisms
Specific immunity, based on antibodies and specifically reactive T lymphocytes, is acquired in a process of immune system stimulation by the corresponding microbial antigens. Humoral immunity is based on antitoxins, opsonins, microbicidal antibodies, neutralizing antibodies, etc. Cellular immunity is based on cytotoxic T lymphocytes (T killer cells) and T helper cells. See Chapter 2 on the principles of specific immunity.
Defects in Immune Defenses
Hosts with defects in their specific and/or nonspecific immune defenses are prone to infection.
Primary defects. Congenital defects in the complement-dependent phagocytosis system are rare, as are B and T lymphocyte def
& Secondary defects. Such effects are acquired, and they are much more frequent. Examples include malnutrition, very old and very young hosts, metabolic disturbances (diabetes, alcoholism), autoimmune diseases, malignancies (above all lymphomas and leukemias), immune system infections (HIV), severe primary diseases of parenchymatous organs, injury of skin or mucosa, immunosuppressive therapy with corticosteroids, cytostatic and immunosuppressants, and radiotherapy.
One result of progress in modern medicine is that increasing numbers of patients with secondary immune defects are now receiving hospital treatment. Such “problem patients” are frequently infected by opportunistic bacteria that would not present a serious threat to normal immune defenses. Often, the pathogens involved (“problem bacteria”) have developed a resistance to numerous antibiotics, resulting in difficult courses of antibiotic treatment in this patient category.
Normal Flora
Bacteria are the predominant component of the normal flora. They proliferate in varied profusion on the mucosa and most particularly in the gastrointestinal tract, where over 400 different species have been counted to date. The count of bacteria per gram of intestinal content is 101–105 in the duodenum, 103–107 in the small intestine, and 1010–1012 in the colon. Over 99% of the normal mucosal flora are obligate anaerobes, dominated by the Gram-neg. anaerobes. Although life is possible without normal flora (e.g., pathogen-free experimental animals), commensals certainly benefit their hosts. One way they do so is when organisms of the normal flora manage to penetrate into the host through microtraumas, resulting in a continuous stimulation of the immune system. Commensals also compete for living space with overtly pathogenic species, a function known as colonization resistance. On the other hand, a potentially harmful effect of the normal flora is that they can also cause infections in immunocompromised individuals.
General Epidemiology
Epidemiology investigates the distribution of diseases, their physiological variables and social consequences in human populations, and the factors that influence disease distribution (World Health Organization [WHO] definition). The field covered by this discipline can thus be defined as medical problems involving large collectives. The rule of thumb on infectious diseases is that their characteristic spread depends on the virulence of the pathogen involved, the susceptibility of the threatened host species population, and environmental factors. Table 1.8 provides brief definitions of the most important epidemiological terms.
Transmission, Sources of Infection
Transmission
Pathogens can be transmitted from a source of infection by direct contact or indirectly. Table 1.9 lists the different direct and indirect transmission pathways of pathogenic microorganisms.
Person-to-person transmission constitutes a homologous chain of infection. The infections involved are called anthropometers. In cases in which the pathogen is transmitted to humans from other vertebrates (and occasionally the other way around) we have a heterologous chain of infection, and the infections are known as zoonoses (WHO definition)
Sources of Infection
Every infection has a source (Table 1.11). The primary source of infection is defined as the location at which the pathogen is present and reproduces. Secondary sources of infection are inanimate objects, materials, or third persons contributing to transmission of pathogens from the primary source to disposed persons.
The Fight against Infectious Diseases
Legislation
on fronting and preventing infectious diseases can sometimes involve substantial incursions into the private sphere of those involved as well as economic consequences. For these reasons, such measures must be based on effective disease control legislation. In principle, these laws are similar in most countries, although the details vary.
The centerpiece of every disease prevention system is provision for reporting outbreaks. Basically, reporting is initiated at the periphery (individual patients) and moves toward the center of the system. Urgency level classifications of infections and laboratory findings are decided on by regional health centers, which are in turn required to report some diseases to the WHO to obtain a global picture within the shortest possible time.
Concrete countermeasures in the face of an epidemic take the form of prophylactic measures aimed at interrupting the chain of infection.
Exposure Prophylaxis
Exposure prophylaxis begins with isolation of the source of infection, in particular of infected persons, as required for the disease at hand. Quarantine refers to a special form of isolation of healthy first-degree contact persons. These are persons who have been in contact with a source of infection. The quarantine period is equivalent to the incubation period of the infectious disease in question (see International Health Regulations, www.who.int/en/).
Immunization Prophylaxis
Active immunization. In active immunization, the immune system is stimulated by administration of vaccines to develop a disease-specific immunity. Table 1.12 lists the vaccine groups used in active immunization. Table 1.13 shows as an example the vaccination schedule recommended by the Advisory Committee on Immunization Practices of the USA (www.cdc.gov/nip). Recommended adult immunization schedules by age group and by medical conditions are also available in the National Immunization Program Website mentioned above. The vaccination calendars used in other countries deviate from these proposals in some details. For instance, routine varicella and pneumococcal vaccinations are not obligatory in Germany, Austria, and Switzerland (see www.rki.de). To simplify the application of vaccines, licensed combination vaccines may be used whenever any components of the combination are indicated and the vaccine’s other components are not contraindicated. Providers should consult the manufacturers’ inserts for detailed information.
Passive immunization. This vaccination method involves administration of antibodies produced in a different host. In most cases, homologous (human) hyperimmune sera (obtained from convalescent patients or patients with multiple vaccinations) are used. The passive immunity obtained by this method is limited to a few weeks (or months at most).
Principles of Sterilization and Disinfection
Sterilization is defined as the killing or removal of all microorganisms and viruses from an object or product. Disinfection means rendering an object, the hands or skin free of pathogens. The term asepsis covers all measures aiming to prevent contamination of objects or wounds. Disinfection and sterilization make use of both physical and chemical agents. The killing of microorganisms with these agents is exponential. A measure of the efficacy of this process is the D value (decimal reduction time), which expresses the time required to reduce the organism count by 90%. The sterilization agents of choice are hot air (180 8C, 30 minutes; 160 8C,120 minutes) or saturated water vapor (121 8C,15 minutes, 2.02 !105 Pa; 134 8C, three minutes, 3.03 !105 Pa). Gamma rays or high-energy electrons are used in radio sterilization at a recommended dose level of 2.5! 104 Gy.
Terms and General Introduction
Terms
Sterilization is the killing of all microorganisms and viruses or their complete elimination from a material with the highest possible level of certainty
An object that has been subjected to a sterilization process, then packaged so as to be contamination-proof, is considered sterile.
Disinfection is a specifically targeted antimicrobial treatment with the objective of preventing transmission of certain microorganisms. The purpose of the disinfection procedure is to render an object incapable of spreading infection.
Preservation is a general term for measures taken to prevent microbe caused spoilage of susceptible products (pharmaceuticals, foods).
Decontamination is the removal or count reduction of microorganisms contaminating an object
The objective of aseptic measures and techniques is to prevent microbial contamination of materials or wounds.
In antiseptic measures, chemical agents are used to fight pathogens in or on living tissue, for example in a wound.
The Kinetics of Pathogen Killing
Killing microorganisms with chemical agents or by physical means involves a first-order reaction. This implies that no pathogen-killing method kills off all the microorganisms in the target population all at once and instantaneously. Plotting the killing rate against exposure time in a semilow coordinate system results in a straight-line curve
Sigmoid and asymptotic killing curves are exceptions to the rule of exponential killing rates. The steepness of the killing curves depends on the sensitivity of the microorganisms to the agent as well as on the latter’s effectiveness. The survivor/exposure curve drops at a steeper angle when heat is applied, and at a flatter angle with ionizing radiation or chemical disinfectants. Another contributing factor is the number of microorganisms contaminating a product (i.e., its bioburden): when applied to higher organism concentrations, an antimicrobial agent will require a longer exposure time to achieve the same killing effect.
Standard sterilization methods extend beyond killing all microorganisms on the target objects to project a theoretical reduction of risk, i.e., the number of organisms per sterilized unit should be equal to or less than 10–6
The D value (decimal reduction time), which expresses the time required to reduce the organism count by 90%, is a handy index for killing effectiveness.
The concentration (c) of chemical agents plays a significant role in pathogen-killing kinetics. The relation between exposure time (t) and c is called the dilution coefficient (n): t ! cn = constant. Each agent has a characteristic coefficient n, for instance five for phenol, which means when c is halved the exposure time must be increased by a factor of 32 to achieve the same effect
The temperature coefficient describes the influence of temperature on the effectiveness of chemical agents. The higher the temperature, the stronger the effect, i.e., the exposure time required to achieve the same effect is reduced. The coefficient of temperature must be determined experimentally for each combination of antimicrobial agent and pathogen species.
Mechanisms of Action
When microorganisms are killed by heat, their proteins (enzymes) are irreversibly denatured. Ionizing radiation results in the formation of reactive groups that contribute to chemical reactions affecting DNA and proteins. Exposure to UV light results in structural changes in DNA (thymine dimers) that prevent it from replicating. This damage can be repaired to a certain extent by light (photoreactivation). Most chemical agents (alcohols, phenols, aldehydes, heavy metals, oxidants) denature proteins irreversibly. Surfactant compounds (amphoteric and cationic) attack the cytoplasmic membrane. Acridine derivatives bind to DNA to prevent its replication and function (transcription).
Physical Methods of Sterilization and Disinfection
Heat
The application of heat is a simple, cheap and effective method of killing pathogens. Methods of heat application vary according to the specific application
Pasteurization. This is the antimicrobial treatment used for foods in liquid form (milk):
High-temperature pasteurization: brief (seconds) of exposure to 80–85 8C in continuous operation
Up erization: heating to 150 8C for 2.5 seconds in a pressurized container using steam injection.
Disinfection. Application of temperatures below what would be required for sterilization. Important: boiling medical instruments, needles, syringes, etc. does not constitute sterilization! Many bacterial spores are not killed by this method.
Dry heat sterilization. The guideline values for hot-air sterilizers are as follows: 180 8C for 30 minutes,160 8C for 120 minutes, whereby the objects to be sterilized must themselves reach these temperatures for the entire prescribed period
Moist heat sterilization. Autoclaves charged with saturated, pressurized steam are used for this purpose:
121 8C, 15 minutes, one atmosphere of pressure (total: 202 kPa).
134 8C, three minutes, two atmospheres of pressure (total: 303 kPa).
In practical operation, the heating and equilibrating hiatus and equalizing times must be added to these, i.e., the time required for the temperature in the most inaccessible part of the item(s) to be sterilized to reach sterilization level. When sterilizing liquids, a cooling time is also required to avoid boiling point retardation.
The significant heat energy content of steam, which is transferred to the cooler sterilization items when the steam condenses on them, explains why it is such an effective pathogen killer. In addition, the proteins of microorganisms are much more readily denatured in a moist environment than under dry conditions.
Radiation
Nonionizing radiation. Ultra-violet (UV) rays (280–200 nm) are a type of nonionizing radiation that is rapidly absorbed by a variety of materials. UV rays are therefore used only to reduce airborne pathogen counts (surgical theaters, filling equipment) and for disinfection of smooth surfaces.
Ionizing radiation. Two types are used: — Gamma radiation consists of electromagnetic waves produced by nuclear disintegration (e.g., of radioisotope 60Co). — Corpuscular radiation consists of electrons produced in generators and accelerated to raise their energy level.
Radio sterilization equipment is expensive. On a large scale, such systems are used only to sterilize bandages, suture material, plastic medical items, and heat-sensitive pharmaceuticals. The required dose depends on the level of product contamination (bioburden) and on how sensitive the contaminating microbes are to the radiation. As a rule, a dose of 2.5! 104 Gy (Gray) is considered sufficient
One Gy is defined as absorption of the energy quantum one joule (J) per kg.
Filtration
Liquids and gases can also be sterilized by filtration. Most of the available filters catch only bacteria and fungi, but with ultrafine filters viruses and even large molecules can be filtered out as well. With membrane filters, retention takes place through small pores. The best-known type is the membrane filter made of organic colloids (e.g., cellulose ester). These materials can be processed to produce thin filter layers with gauged and calibrated pore sizes. In conventional depth filters, liquids are put through a layer of fibrous material (e.g., asbestos). The effectiveness of this type of filter is due largely to the principle of adsorption. Because of possible toxic side effects, they are now practically obsolete.
Chemical Methods of Sterilization and Disinfection
Ethylene oxide. This highly reactive gas (C2H4O) is flammable, toxic, and a strong mucosal irritant. Ethylene oxide can be used for sterilization at low temperatures (20–60 8C). The gas has a high penetration capacity and can even get through some plastic foils. One drawback is that this gas cannot kill dried microorganisms and requires a relative humidity level of 40– 90% in the sterilizing chamber. Ethylene oxide goes into solution in plastics, rubber, and similar materials, therefore sterilized items must be allowed to stand for a longer period to ensure complete desorption.
Aldehydes. Formaldehyde (HCHO) is the most important aldehyde. It can be used in a special apparatus for gas sterilization. Its main use, however, is in disinfection. Formaldehyde is a water-soluble gas. Formalin is a 35% solution of this gas in water. Formaldehyde irritates mucosa; skin contact may result in inflammations or allergic eczemas. Formaldehyde is a broad-spectrum germicide for bacteria, fungi, and viruses. At higher concentrations, spores are killed as well. This substance is used to disinfect surfaces and objects in 0.5–5% solutions. In the past, it was commonly used in gaseous form to disinfect the air inside rooms (5 g/m3). The mechanism of action of formaldehyde is based on protein denaturation.
Alcohols. The types of alcohol used in disinfection are ethanol (80%), propanol (60%), and isopropanol (70%). Alcohols are quite effective against bacteria and fungi, less so against viruses. They do not kill bacterial spores. Due to their rapid action and good skin penetration, the main areas of application of alcohols are surgical and hygienic disinfection of the skin and hands. One disadvantage is that their effect is not long-lasting (no depot effect). Alcohols denature proteins.
Phenols. Lister was the first to use phenol (carbolic acid) in medical applications. Today, phenol derivatives substituted with organic groups and/or halogens (alkylated, arylated, and halogenated phenols), are widely used. One common feature of phenolic substances is their weak performance against spores and viruses. Phenols denature proteins. They bind to organic materials to a moderate degree only, making them suitable for disinfection of excreted materials.
Halogens. Chlorine, iodine, and derivatives of these halogens are suitable for use as disinfectants. Chlorine and iodine show a generalized microbicidal effect and also kill spores.
Chlorine denatures proteins by binding to free amino groups; hypochlorous acid (Holch), on the other hand, is produced in disintegrates into HCl and 1/2 O2 and thus acts as a powerful oxidant. Chlorine is used to disinfect drinking water and swimming-pool water (up to 0.5 mg/l). Calcium hypochlorite (chlorinated lime) can be used in nonspecific disinfection of excretions. Chloramines are organic chlorine compounds that split off chlorine in aqueous solutions. They are used in cleaning and washing products and to disinfect excretions.
Iodine has qualities similar to those of chlorine. The most important iodine preparations are the solutions of iodine and potassium iodide in alcohol (tincture of iodine) used to disinfect skin and small wounds. Iodophors are complexes of iodine and surfactants (e.g., polyvinyl pyrrolidone). While ionophores are less irritant to the skin than pure iodine, they are also less effective as germicides.
Oxidants. This group includes ozone, hydrogen peroxide, potassium permanganate, and peracetic acid. Their relevant chemical activity is based on the splitting off of oxygen. Most are used as mild antiseptics to disinfect mucosa, skin, or wounds.
Surfactants. These substances (also known as surface-active agents, tenses, or detergents) include anionic, cationic, amphoteric, and nonionic detergent compounds, of which the cationic and amphoteric types are the most effective
The bactericidal effect of these substances is only moderate. They have no effect at all on tuberculosis bacteria (with the exception of impotencies), spores, or nonencapsulated viruses. Their efficacy is good against Gram-positive bacteria, but less so against Gram-negative rods. Their advantages include low toxicity levels, lack of odor, good skin tolerance, and a cleaning effect.
Practical Disinfection
Alcohols are therefore often combined with other disinfectants (e.g., quaternary ammonium compounds). Ionophores are also used for this purpose
creations' (feces, sputum, urine, etc.). It is not necessary to kill spores in such applications. Phenolic preparations are therefore frequently used. Contaminated hospital sewage can also be thermally disinfected (80–100 8C) if necessary.
Surface disinfection is an important part of hospital hygiene. A combination of cleaning and disinfection is very effective. Suitable agents include aldehyde and phenol derivatives combined with surfactants.
Instrument disinfection is used only for instruments that do not cause inj ries to skin or mucosa (e.g., dental instruments for work on hard tooth substance). The preparations used should also have a cleaning e
Laundry disinfection can be done by chemical means or in combination with heat treatment. The substances used include derivatives of phenols, aldehydes and chlorine as well as surfactant compounds. Disinfection should preferably take place during washing.
Chlorine is the agent of choice for disinfection of drinking water and swimming-pool water. It is easily dosed, acts quickly, and has a broad disinfectant range. The recommended concentration level for drinking water is 0.1–0.3 mg/l and for swimming-pool water 0.5 mg/l.
Final room disinfection is the procedure carried out after hospital care of an infection patient is completed and is applied to a room and all of its furnishings. Evaporation or atomization of formaldehyde (5 g/m3), which used to be the preferred method, requires an exposure period of six hours. This procedure is now being superseded by methods involving surface and spray disinfection with products containing formaldehyde.
Hospital disinfection is an important tool in the prevention of cross-infections among hospital patients. The procedure must be set out in written form for each specific cash.
.png)
