Ecological changes, including global climate change and deforestation, with farmers and their animals exposed to new arthropods, floods, and drought Microbial evolution whether related to indiscriminate use of anti-infective ...

Some factors that increase emergence or reemergence of infectious pathogens include: ++ Human and animal demographics and population movement with intrusion into new habitats (particularly tropical forests) Irrigation, especially primitive irrigation systems, which fail to control arthropods and enteric organisms Uncontrolled urbanization, with vector populations breeding in stagnant water Increased international commerce and travel with contact or transport of vectors and pathogens (globalization) Breakdown in public health measures, including sanitation, vector control, immunization programs related to social unrest, civil wars, and major natural disasters

+++ EMERGING INFECTIOUS DISEASES ++ An emerging disease is an infectious disease whose incidence has increased in the past two decades and/or that threatens to increase soon. Emerging infectious diseases reflect the arrival of a new pathogen (newly emerging) or an old pathogen that is increasing in incidence, clinical or laboratory characteristics, or geographic range (re-emerging or resurging). An unusual third group is “deliberately emerging” infections, such as anthrax bioterrorism. The appearance of novel coronaviruses (eg, the severe acute respiratory syndrome [SARS] coronavirus and now SARS-CoV-2 [the cause of COVID-19]) are examples of new pathogens, multidrug-resistant Mycobacterium tuberculosis represents an old pathogen with new characteristics, and cholera and Zika in the Americas are examples of old pathogens with a new geographic range (Asia to South America). New methods of detection (eg, molecular) and surveillance (eg, global) have greatly improved our ability to detect and characterize emerging and reemerging infectious diseases. The fundamental methodologies of molecular epidemiology are described in Chapter 4, and their specific applications are discussed in many other chapters throughout this book.

Epidemiology is the study of the distribution and determinants of disease, both infectious and noninfectious, and other perturbations in health

Predictive value of a test is determined by its sensitivity and specificity and the prevalence of disease in a population or the likelihood thereof in a patient based on the history, clinical findings, and epidemiology of the infectious disease agent being considered. The more sensitive a test, the greater its negative predictive value (NPV), thus a patient with a negative test is very unlikely to have the disease. A positive result with a more specific test makes a diagnosis more likely or has a higher positive predictive value (PPV) and basically confirms an etiologic diagnosis. When the prevalence of a disease is exceedingly low or the likelihood is virtually nil based on the history, clinical findings, and epidemiology, even tests with high sensitivity and specificity may have a low PPV. This reality highlights the ...

Behind every clinical specimen submitted to the diagnostic laboratory should be a question. Does my patient have, can I exclude, does the result confirm the disease? Answers to such questions depend on understanding, whether articulated specifically or not, the characteristics of the tests ordered and performed. These characteristics are sensitivity (the test’s ability to rule out [snout] a disease because there are few false-negative results and thus fewer cases missed) and specificity (the test’s ability to rule in [spin] or confirm an etiology because there are few false-positive results). Ideally, a test would have both excellent sensitivity and specificity, but traditional methods often involved a trade-off between the two, which only emphasizes the need to know the clinical question or reason for ordering a test. Molecular methods, however, tend to have improved sensitivity as well as specificity, which is dramatically so for viral etiologic diagnoses.

The diagnosis of a microbial infection begins with an assessment of the clinical and epidemiologic features and formulation of a diagnostic hypothesis. Anatomic localization of the infection depends on physical and radiologic findings (eg, right lower lobe pneumonia, subphrenic abscess). This clinical diagnosis suggests a number of possible etiologic agents based on knowledge of infectious syndromes and their courses. The specific cause or etiologic diagnosis is then established by the application of methods described in this chapter. A combination of science and art on the part of both the clinician and laboratory worker is required: The clinician must select the appropriate tests and specimens to be processed and, where appropriate, suggest the suspected etiologic agents to the laboratory. The laboratory scientist must use the methods that will demonstrate the probable agents and be prepared to explore other possibilities suggested by the clinical situation or by the findings of the laboratory examinations. The best results are obtained when communication between the clinician and laboratory is optimal. ++ ❋ Clinical diagnosis guides approach to etiologic diagnosis

. Antiseptics are disinfecting agents that can be used on body surfaces, such as the skin or vaginal tract, to reduce the numbers of pathogenic agents in the local microbiota.

Disinfection is a less precise term. It implies the destruction of pathogenic microorganisms by processes that fail to meet the criteria for sterilization

Pasteurization is the use of heat at a temperature sufficient to inactivate important pathogenic organisms in liquids such as water or milk, but at a temperature lower than that needed to ensure sterilization.

Absence of growth does not necessarily indicate sterility ++ Sterilization is an absolute term. It means complete killing, or removal, of all living organisms from a particular location or material. It can be accomplished by incineration, nondestructive heat treatment, certain gases, exposure to ionizing radiation, some liquid chemicals, and filtration

+++ DEFINITIONS ++ Death/killing as it relates to microbial organisms is defined in terms of how we detect them in culture. Operationally, it is a loss of ability to multiply under any known conditions

Operationally,

+++ INTRODUCTION ++ From the time of debates about the germ theory of disease, killing microbes before they reach patients has been a major strategy for preventing infection. In fact, Ignaz Semmelweis successfully applied disinfection principles decades before bacteria were first isolated. This chapter discusses the most important methods used for this purpose in modern medical practice. Understanding how these methods work has become of increasing importance in an environment that includes immunocompromised patients, transplantation, indwelling devices, and Covid-19.

FIGURE 2–1. Human blood cells. Stem cells in the bone marrow divide to form two blood cell lineages: (1) the lymphoid stem cell gives rise to B cells that become antibody-secreting plasma cells, T cells that become activated T cells, and natural killer cells. (2) The common myeloid progenitor cell gives rise to granulocytes and monocytes that give rise to macrophages and dendritic cells. (Reproduced with permission from Willey JM: Prescott, Harley, & Klein’s Microbiology, 7th ed. New York, NY: McGraw Hill; 2008.)

pathogen is evading or confounding the immune system. ++ The immune response to infection is presented as two major components—innate immunity and adaptive immunity. The primary effectors of both are cells that are members of the white blood cell series derived from hematopoietic stem cells in the bone marrow (Figure 2–1). Innate immunity includes the role of physical, cellular, and chemical systems that are in place and that respond to all aspects of “foreignness.” These include mucosal barriers, phagocytic cells, and the action of circulating glycoproteins such as complement. The adaptive side is sometimes called specific immunity because it has the ability to develop new responses that are highly specific to molecular components of infectious agents, called antigens. These encounters trigger the development of new cellular responses and production of circulating antibodies, which have a component of memory if the invader returns. Artificially creating this memory is, of course, the goal of vaccines. ++ FIGURE 2–1.

This chapter is not intended to fulfill that function, or, indeed, to be a shortened but comprehensive version of those sources. It is included as an overview of aspects related to infection for other students and as an internal reference for topics that reappear in later pages of this book. These include some of the greatest successes of medical science. The early and continuing development of vaccines that prevent and potentially eliminate diseases is but one example. In addition, knowledge of the immune response to infection is integral to understanding the pathogenesis of infectious diseases. It turns out that one of the main attributes of a successful pathogen is evading or confounding the immune system.

The “maladies” Metchnikoff and the other pioneers of immunology were fighting were infections and, for decades, their field was defined in terms of the immune response to infection. We now understand that the immune system is as much a part of everyday human biologic function as the cardiovascular or renal systems

Finally, our current COVID-19 pandemic caused by the emergence of a new member of the well-known Coronavirus genus threatens to become the leading killer, not just in a century but ever. For students of medicine, understanding the fundamental basis of infectious diseases has more relevance than ever.

Who could have guessed that Helicobacter pylori, not even mentioned in the first edition of this book (1984), would be the major cause of gastric and duodenal ulcers and an officially declared carcinogen?

A new uneasiness that is part evolutionary, part discovery, and part diabolic has taken hold. Infectious agents once conquered have shown resistance to established therapy, such as multiresistant Mycobacterium tuberculosis, and diseases, such as acquired immunodeficiency syndrome (AIDS), have emerged. The spectrum of infection has widened, with discoveries that organisms earlier thought to be harmless can cause disease under certain circumstances.

In the 20th century, advances in public sanitation and the development of vaccines and antimicrobial agents changed this (Figure 1–1), but only for the nations that can afford these interventions. As we move through the second decade of the 21st century, the world is divided into countries in which heart attacks, cancer, and stroke have surpassed infection as causes of premature death and those in which infection is still the leader. That is, unless there is a pandemic causing infection to again become the leading killer everywhere.

When Sir William Osler, the great physician/humanist, wrote these words, fever (infection) was indeed the scourge of the world. Tuberculosis and other forms of pulmonary infection were the leading causes of premature death among the well-to-do and the less fortunate. The terror was due to the fact that, although some of the causes of infection were being discovered, little could be done to prevent or alter the course of disease.

Humanity has but three great enemies: fever, famine, and war

The proton motive force is an electrochemical gradient with two components: a difference in pH (hydrogen ion concentration) and a difference in ionic charge. The charge on the outside of the bacterial membrane is more positive than the charge on the inside, and the difference in charge contributes to the free energy released when a proton enters the cytoplasm from outside the membrane

The free energy may be used to move the cell, to maintain ionic or molecular gradients across the membrane, to synthesize anhydride bonds in ATP, or for a combination of these purposes. Alternatively, cells given a source of ATP may use its anhydride bond energy to create the proton motive force that in turn may be used to move the cell and to maintain chemical gradients.

n eukaryotes, the membrane may be part of the mitochondrion or the chloroplast. In prokaryotes, the membrane is the cytoplasmic membrane of the cell.

For the most part, the organic matter is in macromolecules formed by the introduction of anhydride bonds between building blocks. Synthesis of the anhydride bonds requires chemical energy, which is provided by the two phosphodiester bonds in adenosine triphosphate (ATP; see Chapter 6). Additional energy required to maintain a relatively constant cytoplasmic composition during growth in a range of extracellular chemical environments is derived from the proton motive force. The proton motive force is the potential energy that can be derived by passage of a proton across a membrane

Most of the dry weight of microorganisms is organic matter containing the elements carbon, hydrogen, nitrogen, oxygen, phosphorus, and sulfur. In addition, inorganic ions such as potassium, sodium, iron, magnesium, calcium, and chloride are required to facilitate enzymatic catalysis and to maintain chemical gradients across the cell membrane.

Factors that must be controlled during growth include the nutrients, pH, temperature, aeration, salt concentration, and ionic strength of the medium

Bacteria divide by binary fission, asexual reproduction where a single cell divides giving rise to two cells. Those two cells give rise to a total of four cells and so on. This process of replication requires the acquisition of elements that make up their chemical composition. Nutrients from the environment provide these elements in metabolically accessible forms. In addition, organisms require metabolic energy to synthesize macromolecules and maintain essential chemical gradients across their membranes.

+++ INTRODUCTION ++ Cultivation is the process of propagating organisms by providing the proper environmental conditions. Parasites, bacteria, and viruses all generally require cultivation for detailed study. The field of microbiology has the greatest experience in the cultivation of bacteria and as such, this is the focus of this chapter.

A. Viable Cell Count ++ The viable cell count (Table 4-1) is typically considered the measure of cell concentration. For this, a 1-mL volume is removed from a bacterial suspension and serially diluted 10-fold followed by plating 0.1-mL aliquots on a suitable agar medium. Each single invisible bacterium (or clump of bacteria) will grow into a visible colony that can be counted (see Chapter 5). For statistical purposes, plates containing between 30 and 300 colonies give the most accurate data. The plate count × the dilution × 10 will give the number of colony forming units (CFU)/mL in the undiluted bacterial suspension. Using this method, dead bacteria within the suspension do not contribute to the final bacterial count.

The Measurement of Microbial Concentrations ++ Microbial concentrations can be measured in terms of cell concentration (the number of viable cells per unit volume of culture) or of biomass concentration (dry weight of cells per unit volume of culture). These two parameters are not always equivalent because the average dry weight of the cell varies at different stages of a culture. Nor are they of equal significance: For example, in studies of microbial genetics and the inactivation of microbes, cell concentration is the significant quantity; in studies on microbial biochemistry or nutrition, biomass concentration is the significant quantity.

+++ THE MEANING OF GROWTH ++ Growth is the orderly increase in the sum of all the components of an organism. The increase in size that results when a cell takes up water or deposits lipid or polysaccharide is not true growth. Cell multiplication is a consequence of binary fission that leads to an increase in the number of single bacteria making up a population, referred to as a culture.

Much of our understanding of microbial physiology has come from the study of isolated cultures grown under optimal conditions in laboratories (nutrient excess). However, most microorganisms compete in the natural environment under nutritional stress. Furthermore, a vacant environmental microbial niche will soon be filled with a different microbiome. In the end, appreciating the complex interactions that ensure the survival of a specific microbiome is a balance between availability of nutrients and physiologic efficiency.

+++ SURVIVAL OF MICROORGANISMS IN THE NATURAL ENVIRONMENT ++ The population of microorganisms in the biosphere remains roughly constant because the growth of microorganisms is balanced by the death of these organisms. The survival of any microbial group within an environmental niche is ultimately influenced by successful competition for nutrients and by maintenance of a pool of all living cells, often composed of human cells and a consortium of different microorganisms (referred to as the microbiome or microbiota). Understanding competition for nutritional resources within a given microenvironment is essential to understanding the growth, survival, and death of bacterial species (also known as physiology).

In a microbiologic context, classification is the categorization of organisms into taxonomic groups. Experimental and observational techniques are required for taxonomic classification. This is because biochemical, physiologic, genetic, and morphologic properties are historically necessary for establishing a taxonomic rank. This area of microbiology is necessarily dynamic as the tools continue to evolve (eg, new methods of microscopy, biochemical analysis, and computational nucleic acid biology). ++ Nomenclature refers to the naming of an organism by an established group ...

Identification schemes are not classification schemes, although there may be some superficial similarity. For example, the popular literature has reported Escherichia coli as the causative agent of hemolytic uremic syndrome (HUS) in infants. There are hundreds of different strains that are classified as E. coli but only a few that are associated with HUS. These strains can be “identified” from the many other E. coli strains by antibody reactivity with their O-, H-, and K-antigens, as described in Chapter 2 (eg, E. coli O157:H7). However, they are more broadly classified as a member of the family Enterobacteriaceae.

Identification is the practical use of a classification scheme (1) to isolate and distinguish specific organisms among the mix of complex microbial flora, (2) to verify the authenticity or special properties of a culture in a clinical setting, and (3) to isolate the causative agent of a disease. The latter may lead to the selection of specific pharmacologic treatments directed toward their eradication, a vaccine mitigating their pathology, or a public health measure (eg, handwashing) that prevents further transmission.

Identification, classification, and nomenclature are three separate but interrelated areas of bacterial taxonomy. Each area is critical to the ultimate goal of accurately studying the infectious diseases and precisely communicating these to others in the field.

+++ TAXONOMY—THE VOCABULARY OF MEDICAL MICROBIOLOGY ++ One has only to peruse the table of contents of this book to appreciate the diversity of medical pathogens that are associated with infectious diseases. It has been estimated that we currently have the capacity to identify a surprisingly small number of the pathogens responsible for causing human disease. In part this is due to our inability to culture or target these organisms using molecular probes. The diversity of even these identifiable pathogens alone is so great that it is important to appreciate the subtleties associated with each infectious agent. The reason for understanding these differences is significant because each infectious agent has specifically adapted to a particular mode(s) of transmission, the capacity to grow in a human host (colonization), and a mechanism(s) to cause disease (pathology). As such, a vocabulary that consistently communicates the unique characteristics of infectious organisms to students, microbiologists, and health care workers is critical to avoid the chaos that would ensue without the organizational guidelines of bacterial taxonomy (Gk. taxon = arrangement; eg, the classification of organisms in an ordered system that indicates a natural relationship).

B. Phase-Contrast Microscope ++ The phase-contrast microscope was developed to improve contrast differences between cells and the surrounding medium, making it possible to see living cells without staining them; with bright-field microscopes, killed and stained preparations must be used. The phase-contrast microscope takes advantage of the fact that light waves passing through transparent objects, such as cells, emerge in different phases depending on the properties of the materials through which they pass. This effect is amplified by a special ring in the objective lens of a phase-contrast microscope, leading to the formation of a dark image on a light background (Figure 2-1).

With this microscope, specimens are rendered visible because of the differences in contrast between them and the surrounding medium. Many bacteria are difficult to see well because of their lack of contrast with the surrounding medium. Dyes (stains) can be used to stain cells or their organelles and increase their contrast so that they can be more easily seen in the bright-field microscope.

The bright-field microscope is the most commonly used in microbiology courses and consists of two series of lenses (objective and ocular lens), which function together to resolve the image. These microscopes generally employ a 100-power objective lens with a 10-power ocular lens, thus magnifying the specimen 1000 times. Particles 0.2 µm in diameter are therefore magnified to about 0.2 mm and so become clearly visible. Further magnification would give no greater resolution of detail and would reduce the visible area (field).

Several types of light microscopes, which are commonly used in microbiology, are discussed as follows. +++ A. Bright-Field Microscope

+++ OPTICAL METHODS +++ The Light Microscope ++ The resolving power of the light microscope under ideal conditions is about half the wavelength of the light being used. (Resolving power is the distance that must separate two point sources of light if they are to be seen as two distinct images.) With yellow light of a wavelength of 0.4 µm, the smallest separable diameters are thus about 0.2 µm (ie, one-third the width of a typical prokaryotic cell). The useful magnification of a microscope is the magnification that makes visible the smallest resolvable particles.

Historically, the microscope first revealed the presence of bacteria and later the secrets of cell structure. Today, it remains a powerful tool in cell biology.

Prediction, the practical outgrowth of science, is a product created by a blend of technique and theory. Biochemistry, molecular biology, and genetics provide the tools required for analysis of microorganisms. Microbiology, in turn, extends the horizons of these scientific disciplines. A biologist might describe such an exchange as mutualism, that is, one that benefits all contributing parties. Lichens are an example of microbial mutualism. Lichens consist of a fungus and phototropic partner, either an alga (a eukaryote) or a cyanobacterium (a prokaryote) (Figure 1-1). The phototropic component is the primary producer, and the fungus provides the phototroph with an anchor and protection from the elements. In biology, mutualism is called symbiosis, a continuing association of different organisms. If the exchange operates primarily to the benefit of one party, the association is described as parasitism, a relationship in which ...

Nowhere is biologic diversity demonstrated more dramatically than by microorganisms, cells, or viruses that are not directly visible to the unaided eye. In form and function, be it biochemical property or genetic mechanism, analysis of microorganisms takes us to the limits of biologic understanding. Thus, the need for originality—one test of the merit of a scientific hypothesis—can be fully met in microbiology. A useful hypothesis should provide a basis for generalization, and microbial diversity provides an arena in which this challenge is ever present.

Humans also have an intimate relationship with microorganisms; 50–60% of the cells in our bodies are microbes (see Chapter 10). The bacteria present in the average human gut weigh about 1 kg, and a human adult will excrete his or her own weight in fecal bacteria each year. The number of genes contained within this gut flora outnumber that contained within our genome by 150-fold; even in our own genome, 8% of the DNA is derived from remnants of viral genomes.

. They are responsible for cycling the chemical elements essential for life, including carbon, nitrogen, sulfur, hydrogen, and oxygen; more photosynthesis is carried out by microorganisms than by green plants. Furthermore, there are 100 million times as many bacteria in the oceans (13 × 1028) as there are stars in the known universe. The rate of viral infections in the oceans is about 1 × 1023 infections per second, and these infections remove 20–40% of all bacterial cells each day. It has been estimated that 5 × 1030 microbial cells exist on earth; excluding cellulose, these cells constitute about 90% of the biomass of the entire biosphere.

Microbiology is the study of microorganisms, a large and diverse group of microscopic organisms that exist as single cells or cell clusters; it also includes viruses, which are microscopic but not cellular. Microorganisms have a tremendous impact on all life and the physical and chemical makeup of our planet.