BACTERIOLOGY

Bacteriology is a branch of basic microbiology that is concerned with the diagnosis, prevention and treatment of infectious diseases caused by bacteria. It is simply the study of bacteria that are of medical importance i.e. that cause diseases in human beings and animals. Medical bacteriology is the study of the interactions between pathogenic bacteria and the human body as well as that of other animals and mammals that culminate in the development of infectious diseases in them. It also encompasses the pathogenicity, virulence and laboratory detection as well as the control and the prevention of the diseases caused by the pathogenic bacteria.

Bacteriology is defined as the study of bacteria. Medical Bacteriology is a branch of medical microbiology that is concerned with the diagnosis, prevention and treatment of infectious diseases caused by bacterial pathogens. It is simply the study of bacteria that are of medical importance i.e. that cause diseases in human beings and animals. Medical bacteriology is the study of the interactions between pathogenic bacteria and the human body as well as that of other animals and mammals that culminate in the development of infectious diseases in them. It also encompasses the pathogenicity, virulence and laboratory detection as well as the control and the prevention of the diseases caused by the pathogenic bacteria. As will be seen later in this textbook, there are plethora of bacteria that cause different diseases and infections in humans.

Microbiologists study bacteria as well as other microbes in order to gain basic understanding about their physiology, reproduction and metabolic activities so that they can understand the diseases they cause and develop practicable ways to control them, thereby preventing their emergence and spread within a given population. Prior to the discovery of antimicrobial agents (antibiotics in particular), pathogenic bacteria have caused many deaths and infections in human populations. Some bacterial diseases such as plague, tuberculosis (TB), diarrhea, dysentery, pneumonia, diphtheria, cholera and typhoid fever amongst others used to kill mankind in their thousands and millions. But due to the advent of antibiotics for the therapeutic management of these diseases coupled with improvement in water supply and environmental sanitation, humanity started to witness a drastic fall in some of these world epidemic diseases. However, some of these diseases (e.g. TB) which were once taught to be conquered by man have now re-emerged in the form of multidrug resistant strains, and some have caused some appreciable number of morbidity and mortality across the world.

Today, there is plethora of multidrug resistant strains of some pathogenic bacteria including Staphylococci, Escherichia, Klebsiella, and Pseudomonas amongst others which are defiant even in the face of antimicrobial onslaught; and a handful of these organisms are responsible for a majority of nosocomial infections and even community-acquired infections. About one-third of all deaths in the world today is caused by infectious agents including pathogenic bacteria, and the situation is worse in developing countries where environmental sanitation and access to quality healthcare services are still poor amongst the population. Aside this, a fall in the body’s natural defense against infectious diseases (i.e. the host’s immune system) which could be due to malnutrition and other predisposing factors can also increase people’s chances of becoming infected by pathogenic bacteria. In this chapter, some of the clinically important bacteria will be discussed from the point of view of their pathogenicity and/or host-parasite relationship, laboratory detection, immunity and their treatment in the face of infection.

Microbiologists study bacteria as well as other microorganisms in order to gain basic understanding about their physiology, reproduction and metabolic activities so that they can understand the diseases they cause and develop practicable ways to control them, thereby preventing their emergence and spread within a given population. Prior to the discovery of antimicrobial agents (antibiotics in particular), pathogenic bacteria have caused many deaths and infections in human populations. Some bacterial diseases such as plague, tuberculosis (TB), diarrhea, dysentery, pneumonia, diphtheria, cholera and typhoid fever amongst others used to kill mankind in their thousands and millions. But due to the advent of antibiotics for the therapeutic management of these diseases coupled with improvement in water supply and environmental sanitation, humanity started to witness a drastic fall in some of these world epidemic diseases.

However, some of these diseases (e.g. TB) which were once taught to be conquered by man have now re-emerged in the form of multidrug resistant strains, and some have caused some appreciable number of morbidity and mortality across the world. Today, there is plethora of multidrug resistant strains of some pathogenic bacteria including Staphylococci, Escherichia, Klebsiella, and Pseudomonas amongst others which are defiant even in the face of antimicrobial onslaught; and a handful of these organisms are responsible for a majority of nosocomial infections and even community-acquired infections. Aside this, a fall in the body’s natural defense against infectious diseases (i.e. the host’s immune system) which could be due to malnutrition and other predisposing factors can also increase people’s chances of becoming infected by pathogenic bacteria. The morbidity and mortality caused by pathogenic bacteria in human and animal population has been largely reduced through water purification, immunization (vaccination) and antibiotic treatment.

OVERVIEW OF BACTERIA

Bacteria (singular: Bacterium) is one of the two important members of the prokaryotes (i.e. cells in which the chromosomes are not separated from the cytoplasmic membrane). The second is Archaea. Bacteria are prokaryotic organisms with very simple cell structure. They are single-celled organisms with complex cell wall. Bacterial cells are the simplest possible forms of microorganisms, and they lack mitochondria, Golgi bodies and nuclear membrane. Bacteria are ubiquitous microorganisms with majority of them occurring in the soil, plants, human body, and as free-living organisms in the environment. They are very small and can only be seen with the aid of the microscope. Bacteria are capable of self replication by binary fission (a type of asexual reproduction in which the parent cell splits to produce the daughter cells or offspring) because they contain all the enzymes and biologically active materials required for their growth and self replication. The two basic forms of bacteria based on their cell wall are: Gram-positive bacteria and Gram-negative bacteria.

Bacterial cells that lack cell wall include Mycoplasmas and L-form bacteria. L-form bacteria are forms of bacteria with different shapes. They are sensitive to osmotic shock. L-form bacteria are formed when bacteria including Gram-positive and Gram-negative bacteria go through a period of nutrient depletion or mutational changes. Bacterial cells without cell wall (which is very critical to the survival of a bacterium) usually exist or live inside other cells under controlled osmotic conditions. Bacterial organisms have been implicated in a variety of infectious diseases that occur in man. They cause plethora of economic important diseases in man, livestock and even in animals. Some of these diseases are mild while the others are debilitating and can result to the death of the sufferer. Notably, bubonic plague (popularly known as Black Death) caused by the bacterium Yersinia pestis is one of the bacterial diseases that spread from China through Europe in the 14th century killing millions of people as at the time, and the disease was believed to drastically reduce the world’s population during those periods.

Though, Black Death have been contained and eradicated by mankind; tuberculosis caused by Mycobacterium tuberculosis, bacterial vaginosis caused by Bacteriodes species, typhoid fever, diphtheria and Cholera caused by Vibrio cholerae are amongst the long list of bacterial related infections that are still causing morbidity and mortality in the human race; and these diseases have taken a huge toll on humanity in terms of its devastation. Some bacterial species have also developed resistance to some already available drugs and this has further worsened the plights of sick people as most of these drugs are no longer as effective in the treatment of infections. Despite the growing cases of some non-infectious diseases such as cancer, hypertension, diabetes and stroke, infections and diseases caused by bacteria still accounts for a significant amount of death in the human population.

SHAPES/MORPHOLOGY OF BACTERIA

All bacterial cells are extremely infinitesimal (i.e. microscopic), and are never visible to the naked eyes. Bacteria exist in different sizes and shapes which may range from 0.1 µm to 0.3 µm wide and 1 µm to 10 µm in length depending on whether they are rod or spherical in shape. Bacteria occur in various shapes. However, most bacteria are spherical in shape (cocci) while others are rod-like in shape (bacillus). The bacteria that assume a spherical shape are generally known as cocci (singular: coccus) while those that assume a rod-like shape are known as bacilli (singular: bacillus). However, bacterial cells generally exist in three forms which are spheres, rods and spirals. Figure 1 shows the various shapes in which a bacterial cell can occur.

 

Figure 1: Different Shapes of Bacteria.

BACILLI (RODS): Bacilli (Rod bacteria) may be long or short, thick or slender in form. These features of rod bacteria are seen when they are viewed microscopically. Rod-like bacteria are usually cylindrical in shape, and they may appear as single cells known as bacillus (plural: bacilli) or bacterium. They can also appear as a double cell called diplococci or diplobacterium, and can also appear in chains called streptobacillus or streptobacterium. Coccobacilli are very short rods that can sometimes be mistaken to be cocci (i.e. spherical bacteria). There is also another group of rod-like bacteria known as fusiform bacteria because they have tapered or pointed ends. Rod-like bacteria can also be curved or bent. Some examples of rod-shaped bacteria include: Corynebacterium, Clostridium, Bacillus, Shigella, Escherichia, Salmonella and Listeria.

SPHERICAL BACTERIA: Bacteria that are spherical in shape are generally called cocci (singular: coccus). Spherical bacteria (which can also be known as oval bacteria because they are not always perfect sphere) may be large or small. They can also group themselves in various ways. Cocci can appear as a single cell (coccus), double cell (diplococcus), or combined as four cells (tetracoccus or tetrad). They can also be in chains (streptococcus) or in bulky form or clusters (staphylococcus). Examples of spherical bacteria include: Streptococci, Staphylococci, and Neisseria.

SPIRAL-SHAPED BACTERIA: Spiral-shaped bacteria can also be called Vibrio, and they are generally known to have shape that looks like a screw or a cork. They are known as spirilla (singular: Spirillum) if the cells are rigid and spirochetes if the cells are undulating and more flexible. Spirilla contain one or more loosed turns while spirochetes contain many coils. Examples of spiral-shaped bacteria include: Vibrio cholerae that causes cholera disease, Treponema, Borrelia and Leptospira.

SIMPLIFIED PROGRESSION OF INFECTIOUS AGENTS IN INFECTIOUS DISEASE PROCESSES

To establish an infectious disease, a disease agent (including bacteria, fungi, viruses and protozoa) must first come in contact with a susceptible human host. This phase is called contact or encounter. Humans first encounter with microorganisms starts immediately after birth (period in which the newborn begin to build up its own normal flora) but as the infant grows, he or she can come in contact with varying microorganisms (both harmless and pathogenic organisms) in the environment that are likely to cause an infection. Adults can encounter pathogens from different routes including the air, food, water, fomites or via sexual intercourse. After a successful host-microbe encounter, pathogens begin to find their way into the host’s body via natural openings on the body (e.g. mouth, nose, ears, and vagina) or through wounds, cuts or abrasion on the skin. This phase is called invasion. Microbes can also penetrate deeper tissues of the body via insect bites or through infected sharp objects. Upon entry into the body, the infectious agents begin to find their way to other organs and tissues far away from their point of entry. This phase is called spreading.

Microbes after spreading to their specific and target organs or tissues in the host, must be able to undergo reproduction in order to increase the number of infectious particle to a level that is necessary to establish an infection. This stage is called multiplication. Clinical signs and symptoms of a particular infectious process will not be noticeable until the microbial load in the host has reached the height that is considered necessary to establish a disease condition. It is worthy of note that there can still be some variations in some disease processes where signs and symptoms only appears after a given period of time (i.e. when the infectious agent is still at its window period). Also, some infectious agents produce toxins upon entering their hosts, and in such instances may not necessarily need to undergo multiplication before a disease or infection is established. Infectious agents multiply successfully in the host through a variety of mechanisms including disruption of the host’s immune system, production of toxins and environmental conditions of the body.

Damage of the host’s organ(s) is the next step that follows multiplication of a microbe in vivo. Pathogens have the potential to damage and stop the normal function of one or more organs or tissues of their host, and this leads to a full blown disease. Some disease when not properly treated either therapeutically or via surgery can be wasting i.e. it can be a dead end in which the host eventually dies. If the host survives the pathogen invasion, both host and pathogen can coexist and continue to live together, and the asymptomatic individual can be a reservoir or source via which the microbe can infect a new susceptible host. Host immune response (especially violent responses) to the body’s invasion by a microbe can sometimes lead to catastrophe in which some organs or tissues become affected. This can be seen in autoimmunity, in which the host’s immune response is directed against self or innate antigens of the body. Pathogenic microorganisms that cause diseases in humans may be endogenous (i.e. they are part of the normal flora of the body) or exogenous (i.e. they are microbes that penetrate the body from the outside). Endogenous microorganisms can initiate an infection when the body’s immune system is compromised or when resident microflora becomes transient i.e. moved from its local position to a new location in the body while exogenous microorganisms are often acquired from the external environment such as from food, water, air or from inanimate objects and even from infected individuals.

FEATURES OF BACTERIA THAT ARE PATHOGENS

Bacterial pathogens that cause infections in humans have innate characteristic mechanisms with which they use to suppress the immune response of their host in their bid to establish a disease process. Initiation of an infectious process leads to the development of signs and symptoms in the human host, and these syndromes helps to announce the presence of a bacterium or group of bacteria that are responsible for the disease process. Some of the basic characteristics of pathogenic bacteria are highlighted in this section.

COLONIZATION: Colonization is the first stage of bacterial infection. It is the establishment of a bacterial pathogen at the appropriate site of entry into the host. Portal of entry of a pathogen into the body of a host may include the nose, mouth, eyes, ear, vagina, or broken skin. Bacterial pathogens usually colonize host tissues that are in contact with the external environment, and this makes it easier for the microbe to penetrate host cells and tissues in order to start the process of disease development.

TRANSMISSIBILITY: Pathogenic bacteria have the ability to be transferred from one person to another through direct body contacts, indirect body contacts, and feacal-oral route, through the bite of an insect vector or through airborne means. Fomites and droplet infections can also transfer pathogenic bacteria from one individual to another. Some bacteria species produce spores which allow them to withstand harsh conditions until they find a suitable environment (e.g. the human body) where they can thrive effectively and dissipate their deleterious activities. Other infections can also be acquired by visiting or staying in certain places (e.g. hospital environments where nosocomial bacteria can be picked up). Pathogenic bacteria can also cross the placenta of a pregnant woman and go on to infect the foetus, and they are a source of concern in hospital wards and operating rooms where they cause a range of infectious diseases.

ADHERENCE: Adherence is the process by which pathogenic bacteria attaches itself to the body surfaces of its human host. The word adherence can also be used synonymously with attachment and adhesion. Bacteria produce chemical substances (e.g. adhesins) that allow them to bind specifically or non-specifically to surfaces (both inanimate and animate). Adhesins are macromolecules that bind a bacterium to a specific surface either in vivo or in vitro. The binding of pathogenic bacteria to the cell of a human host is a key requirement for disease development. Adhesion of bacterial cells to implants in the body of a human host (e.g. catheters) promotes the formation of biofilms which is of medical importance. In other words, for a bacterium to be able to initiate a disease process in a human host, it must first of all attach successfully in order to release its virulence factors that promote its injurious effects in the host.

TOXIGENICITY: Toxigenicity is the ability of bacteria to produce toxins that contribute to the development of a disease. Toxins are products of microbes which at low concentrations act on cells or tissues of a host to cause systemic damage. Pathogenic bacteria are known for their ability to produce a wide variety of toxins upon invading a host cell, and these substances helps to increase their pathogenicity and virulence. Toxins, when produced, may be transported by blood and lymph fluids; and they cause cytotoxic effects at tissue sites distant from the original point of invasion or growth of the bacteria producing them. Toxigenicity or toxigenesis is one of the qualities of pathogenic bacteria which help to increase their pathogenicity and virulence. The toxins produced by pathogenic bacteria can be of two types: endotoxin (which are intracellular and are not elaborated by living intact cells) and exotoxin (which are extracellular and are elaborated by living cells). Endotoxins are cell-associated toxins, and they are released by a dead bacterial cell. They are lipopolysaccharides (LPS), and are located on the outer membrane of Gram-negative bacteria. Exotoxins are produced by a living bacterial cell, and they are usually proteins that act enzymatically. Bacteria species that produce toxins include Clostridium, Corynebacterium, Vibrio, Shigella, Bordetella, Escherichia, Pseudomonas, Bacillus and Staphylococcus.

PATHOGENICITY: Pathogenicity is simply defined as the mechanism of infectious disease development caused by a microbe (in this case a pathogenic bacterium) in a human host. It is the structural and biochemical mechanisms whereby pathogenic bacteria cause disease in a human host. Pathogenicity is the ability of a microbe to cause disease in a host. Certain features of pathogenic bacteria that help to enhance pathogenicity include fimbriae, pili, cell wall components, LPS and capsules.

Virulence: Virulence is the severity, degree or intensity of pathogenicity of a microbe. It is usually the combination of invasiveness, toxigenicity and the state of immunity of the host in relation to the infecting bacterial pathogen.

VIRULENCE FACTORS: Virulence factors are microbial products (e.g. toxins) that enhance the degree of pathogenicity of a microbe in a host. A virulent bacterial pathogen causes severe symptoms of a disease than a less virulent microbe in the same individual.

EVASION OF HOST’S IMMUNE SYSTEM: Pathogenic bacteria have intrinsic factors that allow it to evade the immune system of a host. A pathogenic bacterium has the potential to produce disease in a human host, but it will only do so if it has enough virulence factors or power to enter the host cells/tissues and overcome its defense mechanisms. Otherwise, the immune system of the host will defend against the invading bacterial pathogen, thus restoring the body to its normal function.

INVASION: Pathogenic bacteria must be able to enter its host tissues or cells, multiply and spread to other nearby cells in order to properly establish a disease process.

PATHOGENICITY ISLANDS

Pathogenicity islands (PAIs) are the regions of bacterial chromosome (usually of foreign origin) that contain clusters of genes that provoke virulence in the microbe. They are usually acquired by pathogenic bacteria by horizontal gene transfer in the environment; and only pathogenic bacteria harbour PAIs as they are normally absent in non-virulent bacteria of the same species. PAIs are cassettes or clusters of genes that encode a series of virulence factors in pathogenic bacteria; and they are mainly found in the chromosomes of bacteria especially those that are pathogenic (i.e. disease-causing) in nature. Virulence factors in these organisms are usually encoded by the genes found on the pathogenic islands present in the genome of microbes that have them.

PAIs usually contain gene clusters that mediate virulence in a microbe, and they are obtained from other pathogenic bacteria. These virulence gene clusters may be located in either the plasmids or chromosomes of the bacterium that has them, and they are foreign because this group of genes is somewhat different from the overall genetic makeup of the harbouring organism. Because the base sequence of these clusters of genes are different from the rest of the genome and they are only found in some strains of a certain group of bacterial species (i.e. pathogenic bacteria), it is widely believed that pathogenicity islands were acquired and are not actually innate make up of the organisms genome.

Examples of bacteria that have pathogenicity islands in their genome include enteropathogenic Escherichia coli, Salmonella typhimurium, Shigella flexneri and Yersinia species. PAIs encode genes which contribute to the virulence of pathogenic bacteria. Though PAIs are commonly found in Gram-negative bacteria as exemplified above, they have also been reported in some Gram-positive bacteria. Bacterial cells usually have more than one pathogenicity islands, and these gene clusters are very advantageous to bacteria in that they assist in the secretion of certain types of virulent protein molecules that increases their disease mechanism processes in vivo and damage host cells.

These clustered genes for virulence found in the genome of bacteria are usually acquired from other pathogenic organisms in their environment especially through genetic transfer mechanisms such as conjugation and transduction in the form of plasmids or phages. Symbiosis and biodegradation are other functions carried out by bacterial pathogenicity (chromosomal) islands. They generally increase the virulence of bacteria, and are lacking in non-pathogenic strains of organisms in the same genus or species level with those pathogenic bacteria harbouring them.

MICROBIAL ASSOCIATIONS (HOST-PARASITE RELATIONSHIPS)

A host-parasite relationship is an association that exists between two organisms known as the host and the parasite, in which both organisms either derive benefit from the relationship or is harmed in the process. Microorganisms are ubiquitous, and they often exist in association with other forms of life in their ecological niches including man, plants animals and other microbes. The human host for example is regularly in contact with microorganisms. However, only a few of these microbes are able to establish themselves within the host tissues and/or cells to either cause disease or improve the host’s health. To survive and reproduce, the host and the parasite must co-evolve in such a way that their association does not leave any untoward effect on each other. Nevertheless, some host-parasite relationship such as parasitism is only beneficial to the parasite while the host suffers or dies in the association.

The evolution of life on planet earth has allowed both the host and its parasite to co-evolve in several beneficial and disadvantageous relationships. And to survive, the host continually develops novel strategies or mechanisms that protect it from the detrimental activities of the parasite. The co-evolution of the host and the parasite has also allowed the parasite to also develop mechanisms that enable it to survive within the host. Microorganisms in the soil, water, air and in other parts of the environment exhibit or go into a host-parasite association for several reasons including but not limited to the derivation of nutrients, production  of important metabolites or for protection. Bacteria for example are consistently associated with the body surfaces of animals, man and plants. And in the soil, nitrogen-fixing bacteria help in nitrogen fixation – an important growth process required by plants especially leguminous plants.

There are many more bacterial cells on the surface of humans (including the gastrointestinal tract) than there are human cells that make up the body; and these organisms go into association with their human host to either harm them or derive benefit from them. The bacteria and other microbes that are consistently associated with an animal are called the indigenous microbiota or normal microflora of the animal or human. And these microorganisms exhibit a variety of symbiotic interactions including parasitism, commensalism, amensalism, competition, predation, cooperation and mutualism with their animal or human hosts. Amensalism, predation, competition and parasitism are harmful interactions exhibited by microbes while commensalism, cooperation and mutualism are beneficial interactions that microorganisms exhibit in their niche or habitat.

  • SYMBIOSIS: A symbiotic association is a relationship that exists between two or more different organisms. This type of association is often close, and it usually last for a long period of time. Microorganisms and non-microbial organisms can go into a symbiotic association for diverse reasons which can either be beneficial or harmful to any of the organism in the relationship. The symbiotic association of microorganisms (bacteria in particular) with other forms of living organisms is exemplified in various activities of the ecosystem. For example, some bacteria known as nitrogen fixing bacteria (e.g. Rhizobium species) fix nitrogen in symbiotic association in leguminous plants (e.g. beans and peas). This ability of Rhizobium species and other nitrogen fixing bacteria to convert atmospheric nitrogen (N2) to ammonia (NH3) through the process of nitrogen fixation is a critical biological process in the ecosystem because it readily makes N2 to be available in the form in which plants and animals can utilize it. Some microorganisms also go into symbiotic associations with other higher organisms including but not limited to animals, insects, plants and other invertebrates, where they provide a number of beneficial effects to their partners. Bacteria in the stomach of ruminant animals (e.g. cows) help the animals to digest the cellulose component of the plant they feed on, and in the process provide vitamins, proteins and carbon for the ruminant. Beneficial microorganisms (inclusive of bacteria and some fungi) also live in close association with humans, and are found on the skin and in the hosts body where they provide a number of protection against pathogenic organisms as well as help to synthesize vitamins and some growth promoting factors in the body. Symbiotic association includes mutualism, parasitism, competition, predation, cooperation and commensalism.
  • MUTUALISM: Mutualism is a type of association in which both organisms in the relationship benefits from each other. These organisms are generally known as The symbiotic association of fungi and cyanobacteria or algae to form lichens is a typical example of a mutualistic microbial relationship. In a mutualistic associations, two organisms of different species work together, and each benefit from the relationship. Organisms in a mutualistic relationship evolved together. Each of the organisms in mutualistic relationship was part of the other’s environment, so as they adapted to their environment, they made use of each other in a way that benefited both. Mutualism is exhibited by the beneficial bacteria in the gut or gastrointestinal tract of either humans or animals. The beneficial bacteria found in the human or animal gut is also an example of a mutualistic relationship. While the human’s gastrointestinal tract (GIT) or gut cannot digest all of the food that it eats, the beneficial bacteria in the human gut eat the food that the human cannot digest (by breaking it down) and partially digest it, thus allowing the human to finish the job of digestion. The beneficial bacteria benefit by getting food, and the human benefits by being able to digest the food it eats. Mutualism is generally a symbiosis in which both members benefit from the relationship.
  • PARASITISM: Parasitism is a type of relationship in which one partner (known as the parasite) benefits at the expense of the other partner (known as the host) in the association. The term parasite refers to an organism that grows, feeds and is sheltered on or in a different organism while contributing nothing to the survival of its host. Parasitism is a non-mutual symbiotic relationship between species, where one species, the parasite, benefits at the expense of the other, the host. A parasitic relationship is one in which one organism, the parasite, lives off of another organism, the host, harming it and possibly causing death. The parasite lives on or in the body of the host. It is this type of relationship that leads to the establishment of disease or infection in human or animal host. In most of the cases, the host can survive from the infection or disease by using its immune system to restrain the untoward effects of the parasite. But in some cases, the host can die as a result of the deleterious activities of the parasite in the host. Not all parasites have to cause disease. Some parasites such as louse, ticks, fleas, and leeches are parasitic insects or arthropods that do not usually cause disease directly, but they do suck blood from their host including animals and humans. These parasites cause some harm and discomfort to their host during the blood meal. Parasites can also act as vectors (i.e. organisms that transmit disease-causing pathogens to other species of animals and man). Mosquitoes especially the female Anopheles mosquito that harbours the Plasmodium parasites that cause malaria in man are typical examples of vectors because they transmit disease-causing pathogens to their host. The bacteria that cause the bubonic plague (i.e. Yersinia pestis) are carried by rodents, such as rats. The plague bacteria then infect fleas that bite the rats. Infected fleas transmit the bacteria to other animals they bite, including humans. In this case, both the flea and the bacteria are parasites, and the flea is also a vector that transmits the disease causing bacteria ( pestis) from the rat to the human host.
  • COMMENSALISM:Commensalism is an association in which one organism (known as the commensal organism) benefits from the relationship but the other organism neither benefit nor surfer from the alliance. It is symbiotic relationship between two organisms of different species in which one derives some benefit while the other is unaffected. Commensalismis a relationship in which one member benefits, and the other one neither benefits nor is harmed. It is a type of relationships between two organisms where one organism benefits from the other without affecting it. In commensalism, one organism in the association benefits and the other derives neither benefit nor harm from the relationship.
  • Cooperation: Cooperation is a type of symbiosis that benefits both organisms in the relationship. But unlike commensalism, cooperation is not an obligatory type of relationship. In the fungal-plant root interactions (mycorrhizal) for example, normal chlorophyll-containing plants can grow without the help of the fungus; and this shows that the relationship is not obligatory – since some plant can even grow without the support of the fungus. And some fungi, in contrast, do not survive without forming an association with the plant root and vice-versa.
  • AMENSALISM: Amensalism is defined as a relationship in which the product of one microorganism or organism has a negative effect on the survival of another organism. It is a harmful type of association like parasitism in which one partner in the relationship suffers the adverse effect of another organism. Amensalism is an adverse microbe-microbe interaction. In amensalism, one organism produces a substance or compound that has inhibitory effect on the growth of another organism. The secreted compound can even have the ability to kill the other microorganism in the relationship. The phenomenon of amensalism is not only exhibited by microbes. It can also be seen in humans, animals and insects. The molecules secreted by animals and humans as part of their innate immunity (such as those secreted by the phagocytes) have amensalistic effect on the pathogens that invaded the animal or human host; and the human skin also produces substances that limit the growth of pathogenic organisms on the human body. Some microbial products especially those with antimicrobial effect (e.g. antibiotics) can produce amensalistic effects on the organisms they are in a relationship with; and this could lead to the death or inhibition of the growth of the other microbe.
  • COMPETITION: Competition is defined as a microbial interaction between two microbes that are attempting to use the same resources in a given habitat. It arises when two organisms living in a particular population tries to acquire the same resources or nutrients especially those resources that are in short supply in their community. Microbes compete for several factors including but not limited to space, water and nutrients in their environment. And in most of the cases, it is the survival of the fittest that allows the stronger organism to thrive over the less-stronger organism – since the resource(s) they are competing for may be limiting in their environment. The most dominating microbe outcompetes the other organism (the slow-growing microbe); and thus outgrows it since it is now in charge of the limiting nutrient or resource(s) in that particular microbial community.
  • PREDATION: Predation is defined as the phenomenon in which the predator attacks and kills the prey. In predation, the prey is usually smaller and physically less-powerful than the predator; and this natural advantage gives the predator an edge over its prey – which it kills and feeds upon. Though predation may be perceived as a harmful association (one that often leads to the death of the prey), it has several beneficial effects. For example, some predators only ingest their prey to give them protection and thus provide a high supply of nutrients to the ingested prey. Ciliates are protozoan’s that ingest the bacterium, Legionella pneumophila in aquatic environments; and thus protect the bacterium from the harmful effects of chlorine – which is often used to control the bacterium in air-conditioning units and cooling towers.   

NORMAL BACTERIAL FLORA OF MAN

The human body is inundated with plethora of harmless microorganism’s including fungi and bacteria found in different parts of the body (Figure 2). These organisms which are generally known as normal microflora are found in both the external and internal part of the body. The relationship between indigenous microorganisms of humans (i.e. normal microflora) and the human body or host itself is a typical example of a host-parasite relationship or host-microbe association that is usually beneficial to both the host and the microbe in the relationship.

There is usually a state of equilibrium maintained between the host and the microbe; and this balance ensures that the host and the normal microflora coexist together and survive mutually without causing harm or injury to each other. This state of balance between the host and the normal microflora is normally experienced when the human host in particular is in a healthy state. A functional immune system aside other physiological or physical barrier of the body such as an intact skin helps the host to maintain a state of equilibrium that allow both the normal microflora and the host to coexist without the development of an infectious disease.

Disease or infection usually develops when this state of balance or equilibrium in the body is disrupted either by disease development or dilapidated immune system. The development of disease or infection in this case could be caused by endogenous microorganisms – which are microbes innately found in the host such as normal microflora or from exogenous microorganisms – that emanate from the environment or from other infected humans, animals or fomites.

Normal microflora which can also be referred to as flora or normal microbiota is the totality of microorganisms that are inherently present in a particular environment, body or location at every specific point of time. They generally refer to harmless microorganisms that are frequently found in particular anatomical sites of a healthy host including humans and animals. And it is worthy of note that the terms normal microflora, microflora, indigenous flora, normal commensal flora and normal microbiota are used synonymously to mean the same thing.

Resident microflora (autochthonous flora) are those microorganisms which are permanent dwellers or natural inhabitants of a particular environment or body at any given point of time while transient microflora (allochthonous flora) are microorganisms which only have a temporary habitation in an environment or body at any given time. Microorganisms become transient when they move from one part of the body or their natural environment to another. The microorganisms that naturally make up the microbiota of the human body are normally harmless and they pose no potent danger to the health of the individual. These harmless microorganisms play a variety of important role in the entire upkeep and protection of the human body from disease-causing microorganisms (pathogens), and many floras in the digestive system of humans also aid digestion and other internal metabolic activities by secreting substances or enzymes that spur or fasten these processes.

Microbiota which can also be called normal microflora is the totality of microorganisms that are inherently present in a particular environment, body or location at every specific point of time. Mycoflora are fungal organisms that live in particular sites of the body without causing infection or disease. Microbiota goes into competition with pathogens on and in the human body with a view to subverting their pathogenic and virulent activities within the host. They do this through a process called amensalism (a bacterial interference mechanism). In amensalism, normal microflora utilize available space, nutrients and other resources in the host (required by the pathogen to cause infection and disease) and produce substances that resist their disease-causing mechanisms in the host.

Normal flora can also become opportunistic microorganisms which only cause disease in the host by chance i.e. when the environment or body of the individual favors their blossoming (e.g. in an immunocompromised case such as in an AIDS patients or an individual whose immune system has been suppressed due to therapy). The overuse of antibiotics, stress and nutritional imbalance in human beings can cause their microbiota to become pathogenic in nature. The microbiologist should acquaint him or herself on the basic knowledge of the different microbiota that make up different parts of the human body or the environment from which the samples they will be working with in the laboratory are actually made up of. This will allow the scientist to make concrete conclusions on the inferences drawn from a particular research or test so that the final judgment of an experiment is not based on the microbiota (which are not harmful and might not necessarily be the target or cause of the malady being deciphered).

 

Figure 2: Human anatomy showing sites of the body colonized by microorganisms.

 OPPORTUNISTIC PATHOGENS AND INFECTIONS

Opportunistic pathogens are microorganism that normally does not cause disease but nevertheless can cause disease under certain relatively unusual circumstances such as that provided by a weakened or compromised immune system (e.g. in HIV/AIDS infected individuals). They are microorganisms that under normal circumstances in healthy hosts do not cause disease. However, they act as commensals, but when given opportunity due to underlying injury or disease such as immunodeficiency, they can cause disease or infections called opportunistic infections. Opportunistic infections are infections caused by opportunistic pathogens in people with weakened immune system.

Commensals are organisms that are living in close association with another organism of a different species where neither has an obvious effect on the other. They can be referred to as the normal microflora of the body. Commensal microflora (normal microflora, indigenous microbiota) consists of those micro-organisms, which are present on body surfaces covered by epithelial cells and are exposed to the external environment (gastrointestinal and respiratory tract, vagina, and the skin). Opportunistic pathogens often are otherwise benign members of an individual’s normal flora, though they can also be relatively common environmental organisms not found in the human body. They only cause infection in individuals with a weakened or compromised immune system. An opportunistic pathogen basically needs to be in the right place at the right time to cause disease but normally these circumstances do not coincide and the organism consequently is otherwise harmless.

NEISSERIA GONORRHOEAE

Neisseria gonorrhoeae is a Gram-negative, oxidase-positive, non-motile, non-sporulating, non-capsulate, diplococcus found asymptomatically in humans. N. gonorrhoeae is found in the family Neisseriaceae and genus Neisseria which contains two important human pathogens viz: N. gonorrhoeae and N. meningitidis (which causes meningococcal meningitis, an inflammation of the meninges of the brain and spinal cord).  N. gonorrhoeae, a bacterium known to occur in pairs (i.e. diplococcus) can also be called gonococcus. Gonorrhea is usually symptomatic in men and asymptomatic in women. The genitourinary tract, conjunctiva of the eye, pharynx, throat, and the rectum are usually the parts of the body that are affected in N. gonorrhoeae infection. In men, the urethra, epididymis, bladder and prostrate are usually affected by the organism but N. gonorrhoeae colonizes the urethra more than these other parts causing urethritis and dysuria (difficulty in urination).

Image result for Neisseria gonorrhoeae

Illustration of Gram stain of N. gonorrhoeae. N. gonorrhoeae appears as a diplococcus; and the organism can either be intracellularly or extracellularly located in cells such as neutrophils

But in women, N. gonorrhoeae infects the cervix, rectum, urethra and vulva. In women, infertility or ectopic pregnancy (i.e. development of the embryo in the fallopian tube instead of the uterus) can result following N. gonorrhoeae colonization of the pelvis (where it causes pelvic inflammatory disease, PID). Sexual intercourse especially with an infected individual is the primary route via which this pathogen can be acquired and transmitted. The endocervix and urethra are mostly affected in females, but in males the urethra is the most affected organ. Gonorrhea infection in women can also facilitate the easy contraction of HIV due to the inflammation of the genital region which allows the virus to gain entry during sexual intercourse. Ophthalmic neonatorum (blindness in neonates) can also occur in newborn infants whose mother’s vagina is infected with N. gonorrhoeae. The infant’s eye primarily become infected with gonococcus during delivery, and if left untreated the child can go blind.  

PATHOGENESIS OF NEISSERIA GONORRHOEAE INFECTION

Upon invasion, N. gonorrhoeae attaches to the mucosal surfaces of the urethra in males and cervix in female using its pili, fimbriae and other adherence molecules (known as adhesins). This initial attachment sparks up a local inflammatory reaction at the site of invasion, and attachment and penetration of the microbe into the host cell is largely made possible by the action of the pili and adhesins. The most common symptom of gonorrhea in adult male’s aside urethritis and dysuria may include frequent painful urination, and the discharge of scanty, clear or cloudy and purulent fluid. Purulent vaginal discharges are the major symptom of the disease in adult females who are usually covert with the infection. N. gonorrhoeae specifically attaches to the columnar epithelial cells of the urogenital tract with its surface protein, opacity associated protein (Opa) and other adhesins that allows it to do so.

Adhesion of gonococcus to the mucosal surfaces of the genitourinary tract prevents it from being washed away by the flushing action of the urine in males and the natural vaginal discharges in females. Infection with gonococcus is predominantly through a direct and intimate person-to-person sexual contact, and painful urination (in males) and unusual purulent vaginal discharge (in females) are the most important syndromes that follow infectivity. Gonococcus infection is usually limited to its site of entry where it cause local injury and inflammation, and can rarely spread to the blood stream or other vital tissues of the body. Several disseminated bacterial infections including pelvic inflammatory disease (PID), gonorrheal bacteremia, gonorrheal arthritis, and gonorrheal endocarditis can ensue following the spread and dissemination of gonococcus in the body. These later infection are generally referred to as disseminated gonococcal infections (DGIs). Anal intercourse (especially in homosexual males) can also result to a variety of symptomatic rectal infections in persons engaging in such activity.

LABORATORY DIAGNOSIS OF NEISSERIA GONORRHOEAE INFECTION

Centrifuged urine, urethral exudates, and cervical exudates are the main specimens collected in suspected cases of N. gonorrhoeae infection. Rectal swab can also be obtained from patients suspected to have engaged in anal sex, and who may have presented clinical syndromes of rectal infections. Gram smear of genitourinary samples which demonstrates the kidney-bean shape of the microbe (i.e. Gram-negative diplococci within a neutrophil) is predominantly the most important laboratory diagnostic decisive factor for gonorrhea especially in adult males. Gram smear is rarely used to diagnose the disease in infected females due to morphological similarity of other bacteria from the female vagina with gonococcus. Gram smears of urethral exudates on swab sticks should be rolled onto clean glass slide immediately after collection, fixed (using methanol) and stained without any prior immersion in normal saline. Throat swabs can also be obtained from patients engaging in oral sex for culture.

Image result for Neisseria gonorrhoeae

Illustration of the growth of N. gonorrhoeae  on New York City medium agar, which is a selective medium for isolating the organism from clinically important samples

Gonococcus is a very sensitive organism, thus care must be taken in handling specimen, selecting culture media and in analyzing culture results in order to differentiate the pathogen from normal microflora of the body. N. gonorrhoeae produces small, raised, translucent or grey colonies after one or two days of culture or overnight incubation in the presence of CO2 at 35oC. N. gonorrhoeae is a fastidious, aerobic or facultative microbe, and thus requires additional nutrient (e.g. blood and animal protein) for growth in selective or enriched culture media such as the Modified New York City medium. It occurs intracellularly and extracellularly in its host cell, but it is typically seen as diplococcus in pus (polymorphonuclear) cells under the microscope. N. gonorrhoeae grows well on chocolate agar and on other enriched medium including Martin-Lewis medium and Thayer Martin medium (each of which contain antibiotics that inhibit bacteria and fungi) that enhances its growth. Serological investigations including the use of PCR for DNA amplification and DNA probe has also been developed for the laboratory diagnosis of gonococcus infection.

TREATMENT OF NEISSERIA GONORRHOEAE INFECTION

In the past, gonorrhea was successfully treated with penicillin G (which is administered intramuscularly), but the development of resistance mechanisms including β-lactamase enzymes and alteration of N. gonorrhoeae penicillin-binding-proteins (PBPs) due to the widespread use of penicillin has made this antibiotic to be ineffective for treating gonorrhea. Currently, the center for disease control and prevention (CDC) recommends the use of fluoroquinolones (e.g. levofloxacin, ofloxacin and ciprofloxacin) and third-generation cephalosporins (e.g. cefixime, ceftriaxone) which can either be administered orally or intramuscularly for the treatment of gonorrhea. Antibiotics such as azithromycin, erythromycin, or doxycycline are also included in the treatment options for gonococcus infected patients due to the possibility of co-infection with other STIs including syphilis and Chlamydia trachomatis infection. Gonorrhea infection is incomplete if sex partners are left out. Due to the asymptomatic nature of the infection in females, it is critical to refer and treat sex partners of infected individuals in order to avoid the recurrence of the disease in the treated patient.

PREVENTION AND CONTROL OF NEISSERIA GONORRHOEAE INFECTION

Awareness and education of the general public, coupled with proper treatment of infected patients especially those that are covert are very paramount to the control and prevention of N. gonorrhoeae infection. Avoiding multiple sexual partners and remaining faithful to one’s partner are critical in preventing the spread and transmission of the disease. Use of protection (e.g. condoms) during sexual intercourse can also confer some level of protection.

CORYNEBACTERIUM DIPHTHERIAE

Corynebacterium diphtheriae is a Gram-positive, non-spore forming, aerobic, rod-shaped and motile bacterium that causes diphtheria, an upper respiratory tract illness. They are pleomorphic organisms exhibiting different characteristic morphological shapes including V-shapes, irregular shapes and club-shapes. C. diphtheriae and other species in the genera Corynebacteria grow on the mucous membrane of the upper respiratory tract, skin, nares and wounds of humans. C. diphtheriae is an airborne bacterial pathogen that is resistant to drying, and can be transmitted in human populations via nasal secretions. C. diphtheriae is a toxin-producing bacterium that produces diphtheria toxin, an exotoxin that propagates the pathogenicity of the bacterium in vivo.

PATHOGENESIS OF CORYNEBACTERIUM DIPHTHERIAE INFECTION

C. diphtheriae causes both local and systemic infections in humans. Local infections can occur in the tonsils, nose, conjunctiva and the pharynx while systemic infections may affect the kidneys, cardiac muscles of the heart, adrenal glands and the liver cells. Following the invasion of C. diphtheriae into the body through the respiratory route, the bacterium becomes deposited on the local tissues of the throat and tonsils. C. diphtheriae harbours the β prophage that contains the tox gene responsible for the production of diphtheria toxin. β prophage is a temperate bacteriophage, and it is what controls the production of diphtheria toxin by C. diphtheriae. Diphtheria toxin contains two domains: the A and B domains.

After production, diphtheria toxin become absorbed by the damaged mucous membranes of the tonsils and throat, and this leads to destruction of epithelial cells coupled with an inflammatory reaction, all of which culminates to the formation of a grayish-white exudates called pseudomembrane that surrounds the tonsils, larynx and pharynx. Pseudomembrane is a lesion that contains cells of C. diphtheriae and damaged host cells, and the manifestation of pseudomembrane on the tonsils, larynx and pharynx can eventually result in suffocation due to blockage of air passage. Swelling of the lymph nodes in the neck region can also occur, and profuse bleeding can ensue following the clinical attempt to remove the pseudomembrane. Death can occur if left untreated. Clinical signs and symptoms of diphtheria may include fever, cough, and discharge of thick mucopurulent secretions from the nares.

LABORATORY DIAGNOSIS OF CORYNEBACTERIUM DIPHTHERIAE INFECTION

Laboratory diagnosis of C. diphtheriae infection is mainly dependent on identification of the pathogen from clinical specimens including throat and nose swabs. Physical examination of the throat and tonsil regions of infected patients for pseudomembrane appearance also aids diagnosis. Specimens are examined microscopically by Gram staining for the detection of beaded heads of C. diphtheriae containing metachromatic granules. Bacterial culture in Loeffler serum medium and Tellurite blood agar are used for the primary isolation of C. diphtheriae from nose and throat swabs. PCR tests and serological tests including ELISA can also be used to diagnose the infection.   

Image result for Corynebacterium diphtheriae

Illustration of the rod shaped morphology of C. diphtheriae  in a Gram stain. C. diphtheriae forms beaded heads containing metachromatic granules when stained and viewed under the microscope.

C. diphtheriae  growing on blood agar.

TREATMENT OF CORYNEBACTERIUM DIPHTHERIAE INFECTION

The antibiotics used for treating diphtheria include penicillin, erythromycin, clindamycin, gentamicin and vancomycin. Diphtheria antitoxin which counters the effect of diphtheria toxin in vivo can also be used in severe diphtheria infections. Early treatment of diphtheria using antibiotics and antitoxin will help to eliminate and neutralize the toxigenicity of the pathogen.

PREVENTION AND CONTROL OF CORYNEBACTERIUM DIPHTHERIAE INFECTION

The control and prevention of diphtheria is largely dependent on sustained massive immunization of susceptible individuals using the diphtheria, tetanus and pertussis (DTaP) vaccine. This approach will help to maintain a high level of active immunity in terms of herd immunity in a population and drastically reduce the spread and distribution of toxin-producing C. diphtheriae in a particular geographical area.

HAEMOPHILUS INFLUENZAE

Haemophilus influenzae is a small, Gram-negative, non-sporulating, non-motile, urease positive, indole positive, pleomorphic, rod-like or coccobacillus blood-loving bacterium in the family, Pasteurellaceae. H. influenzae, a non-toxin producing bacterium was first isolated during the 1890 influenza pandemic, and it is often referred to as a “blood-loving” bacterium (i.e. haemophilic bacterium) because it requires growth factors which are present in blood for growth. These growth factors are hemin (X factor) and NAD or NADP (V factor), and they play significant role in the growth of H. influenzae.

Haemin is required by H. influenzae to synthesize cytochromes, catalase and peroxidase while nicotinamide adenine dinucleotide (NAD)/nicotinamide adenine dinucleotide phosphate (NADP) is used as an electron carrier by the bacterium in its oxidation-reduction reaction. In particular, H. influenzae type b (Hib), an encapsulated strain is the chief cause of infections in humans especially children where it causes a variety of infections including bacteraemia, pneumonia, meningitis, and epiglottis. In adults, Hib cause cellulitis, emypyema, septic arthritis and other invasive infections. Hib causes the most severe infections in humans.

PATHOGENESIS OF HAEMOPHILUS INFLUENZAE INFECTION

The respiratory tract is often the major route via which Haemophilus influenzae type b (Hib) enters the body and cause infection. Though the pathogenesis of H. influenzae is not completely explicit, the capsular polysaccharide is believed to be the main driving force behind the microbe’s virulence. The capsular polysaccharide confers protection to H. influenzae from lysis by complement molecules and opsonization by phagocytes. Infections with capsulated H. influenzae can occur in children (under the age of 5 years) who lack capsular antibodies which protect the host against Hib infections.

Generally, Hib, the capsulated strain of Haemophilus causes the most life-threatening forms of infections in children below the age of 5 years old especially when it becomes invasive, affecting various tissues and causing illnesses that include bacteraemia, cellulitis, osteomyeltis, epiglottitis, arthritis and meningitis. However, Hib rarely cause disease in adult individuals and children above the age of 5 years. Pneumonia, Otitis media (ear infection), epiglottitis, bacteraemia, and meningitis are some of the infections caused by Hib in infants and children, and these can occasionally be caused by non-typable H. influenzae strains.

LABORATORY DIAGNOSIS OF HAEMOPHILUS INFLUENZAE INFECTION

H. influenzae requires complex nutritional growth requirements for growth (in particular: blood-containing culture media that supplies haemin and NAD/NADP). Sputum, pus, CSF, blood and nasopharyngeal specimens are often the main specimens required for laboratory investigation for H. influenzae infection. Chocolate agar is the best culture media for the growth of H. influenzae in the laboratory. Growth of H. influenzae occurs aerobically with slight CO2 tension (usually 5 %) at 35-37oC. Gram staining of specimens is also very useful when looking out for H. influenzae because smears demonstrate the pleomorphic and thread-like nature of the Gram-negative rods. Dilute carbol fuchsin instead of safranin (the known and widely used counterstain in Gram staining) is used as the secondary (counter) stain when Gram staining samples suspected to harbour H. influenzae. The demonstration of the requirement for V factor on blood agar is confirmatory for the presence of H. influenzae in a specimen. H. influenzae exhibit satelliting effect on blood agar and this phenomenon demonstrates requirement for V factor.

Image result for Haemophilus influenzae

Illustration of Gram stain of a sputum sample showing H. influenzae appearing as a Gram-negative coccobacilli.

Satellitism (satellite phenomenon) is the observable effect seen when certain growth-factor-requiring microbes (such as H. influenzae) can grow efficiently on a growth media which lacks the required growth factor but which supports the growth of another microorganism which can provide the required growth factor. S. aureus (a feeder organism in this case) is used in performing satellitism test because it supplies V factor into the growth medium, and this phenomenon allows satellite growth of colonies of Haemophilus species that requires NAD/NADP for growth. H. influenzae does not cause haemolysis on blood agar. Requirement for haemin (X) is investigated by the porphyrin test, and the presence of H. influenzae in the sample is confirmed if the result of the test is negative because H. influenzae does not synthesize porphyrin and haeme. Only Haemophilus species that do not require X factor for growth are known to be positive for the porphyrin test because such species synthesize haeme and porphyrins. Serological tests can also be employed to detect the serotypes of Haemophilus. H. influenzae is indole and urease positive when subjected to biochemical testing.  

Satellitism test for detection of  H. influenzae

TREATMENT OF HAEMOPHILUS INFLUENZAE INFECTION

The drugs of choice for the treatment of H. influenzae infections include ampicillin, chloramphenicol, sulphamethoxazoles, aminoglycosides, tetracyclines and some third generation cephalosporins including ceftazidime, cefotaxime, augmentin (amoxicillin-clavulanic acid) and ceftriaxone.

PREVENTION AND CONTROL OF HAEMOPHILUS INFLUENZAE INFECTION

Non-typable species of H. influenzae is a normal flora of the upper respiratory tract of humans. Thus, infections are likely to occur from these species when the immune system becomes weakened. However, Hib can be transmitted from infected persons to non-infected individuals through the respiratory tract as aerosols. Approved Hib conjugate vaccines should be administered to children especially those at higher risk of infection, and booster doses should be included following laid down medical guidelines. Proper vaccination of children from the age of 2 months and above against the infection will go a long way in minimizing mortality especially bacterial meningitis which is associated with H. influenzae infection. 

MYCOBACTERIUM TUBERCULOSIS

Mycobacterium tuberculosis is a slim, non-motile, non-spore forming, Gram-positive, obligate aerobe, and acid-fast bacillus (rod) with a waxy cell wall. It is found in the genus Mycobacterium and family Mycobacteriaceae. Aside M. tuberculosis, M. bovis (cattle/animal pathogen), M. avium and M. leprae (causative agent of leprosy/Hansen’s disease) are the other important species of the genus Mycobacterium that causes disease in humans. Other non-tuberculous Mycobacteria are members of the human normal microflora, found in water surfaces, are non-contagious and are generally referred to as atypical Mycobacteria. Consumption of unpasteurized (raw) milk and close contact with infected cattle can cause M. bovis infection in humans.

The cell wall of all bacteria in the Mycobacterium genus is different and very unique in that their cell wall is made up of high concentrations of lipids containing long chain fatty acids known as mycolic acid (which makes the cell surface of the bacterium hydrophobic). M. tuberculosis and other related species in the genus Mycobacterium are mostly called acid-fast bacilli because they show a tendency of acid-fastness upon staining. The reason for their acid-fastness is attributed to the high mycolic (fatty) acid content of their cell wall which makes them difficult to be stained readily; but once stained, Mycobacterium species resist decolourization by alcohol or acid – a phenomenon that aids in their detection and differentiation from other non-mycolic bacteria. M. tuberculosis is an airborne pathogen responsible for causing an infection known as tuberculosis (TB) in humans, and it is one of the leading causes of death in the world.

PATHOGENESIS OF MYCOBACTERIUM TUBERCULOSIS INFECTION

M. tuberculosis is an airborne pathogen, and thus the main route of transmission or acquiring the disease is through the upper respiratory system/airways. An infection with the tubercle bacillus, M. tuberculosis, is usually initiated following an inhalation of microscopic particles in aerosols that originate from an active pulmonary TB disease; and the incubation period of the disease after infection is usually 4-12 weeks. An exposure to M. tuberculosis may lead to infection, but most infections do not lead to a TB disease depending on the immune state of the affected individual. People with an active pulmonary TB disease expel plethora of infectious microscopic tubercle bacilli into the atmosphere when they sneeze, cough, expectorate/spit or speak. Because the infectious dose of TB is very low (about 5-10 bacteria), inhaling as little as 10 bacteria in an aerosol, droplets or dust particles can cause an infection.

The human respiratory system showing portal of entry and possible sites of M. tuberculosis infection in the body.

However, individuals with prolonged, frequent, or intense contact with TB disease persons are at particularly high risk of becoming infected than people with lesser contact. The immune system of the human host and the strain of the tubercle bacilli are critical in determining the pathogenesis of the disease – in that people with active immunity tends to resist and contain the disease more than those with a weakened resistance. After inhalation, the bacterium M. tuberculosis is carried past the upper and middle airways system until it reaches the alveolar surfaces of the lungs where they are deposited. Adjacent lymph nodes are also infected by the bacterium, and small inflammatory lesion is formed around these sites as well. Inside the lungs, alveolar macrophages surround the bacterium by the process of phagocytosis, and this leads to hypersensitivity reaction that forms tubercles (small and hard nodules characteristic of the TB disease). This is known as primary infection, and in this scenario; the host immune system is able to restrict and contain the tubercle bacillus within the pulmonary system– thus preventing it from spreading and leading to a disease state.

At this stage, TB infection is limited, and the lesions formed are self-healing even though all the tubercle bacilli may not be destroyed. People with weakened immune system experience active pulmonary infection, and this leads to the destruction of the lungs and the spread of the pathogen to other parts of the body leading to death. However, most cases of primary TB infections are usually handled well by the host immune system and the Mycobacterium continues to multiply intracellularly (but under the watch of macrophages) without any significant damage to the host cells.

In secondary infection (which may be active pulmonary or extra-pulmonary infection), M. tuberculosis in the lungs becomes reactivated after several months or years due to malnutrition, impaired immunity or poor health condition of the individual. Secondary (post-primary) infection can also occur when primary infections do not heal effectively and this is usually experienced in about 10% of the primary TB cases. Tubercle bacilli that survived the primary lesion or infection processes are mainly responsible for sparking up a post-primary (reactivation) types of TB. The further course of the TB disease (i.e. from primary to secondary infection) depends on the outcome of the encounter between the host’s specific cell-mediated immunity (CMI) and the tubercle bacilli itself.

CMI is usually protective and lifelong in some TB infected individuals. But in some other cases, the tubercle bacilli or particles become dislodged from their containment by the mechanisms of the CMI into the host’s airways later in their lifetime, and this occurs when there is a significant reduction in the individual’s T-cell immune response. At this stage, the pathogen multiplies sporadically in the host’s body, dissemination to vital body begins and the TB disease symptoms start to emanate. The clinical signs and symptoms of TB (i.e. in secondary infection) which only appears in pulmonary TB or extra-pulmonary TB (i.e. when the disease becomes active) and may resemble other lung/respiratory diseases include loss of appetite, chest pain, prolonged and productive coughs with blood, fever, unexplained weight loss, poor growth in children, and fatigue. The disseminated forms of TB which emanate from a secondary infection include military TB (which affect the spleen, lymph glands and liver due to dissemination of the pathogen via blood), tuberculosis meningitis (which affect the brain and meninges), renal and urogenital tuberculosis (which affect the GIT and kidney) and bone and joint tuberculosis (which affect the spinal cord or vertebrae).

DIFFERENCES BETWEEN A TB INFECTION AND A TB DISEASE

It is noteworthy that an infection with M. tuberculosis does not necessarily connote that someone has a TB disease. Exposure to aerosols, dust particles and respiratory droplets (e.g. sputa, sneeze and cough from an infectious TB patient) containing sufficient amount or dosage of tubercle bacilli is the first basis for the acquisition of M. tuberculosis. Whether the exposure that led to an infection will consequently result into a TB disease is dependent on so many factors including the strain of the infecting pathogen, the state of the host’s immunity, host’ health condition amongst others. TB infection and TB disease are quite two different phenomenons.

While the former (i.e. TB infection) presents no clinical symptom, is not infectious, has a normal chest X-ray results and shows a negative sputum smear and culture test results; the latter (i.e. TB disease) presents with clinical symptoms (cough, fever, and weight loss), is infectious, chest X-ray reveals lesions, and sputum smear and culture test results are positive. In both cases of a TB infection and TB disease, the bacterium M. tuberculosis is always present and skin (tuberculin) test results are always positive in these individuals. However, people with a TB infection are not infectious (i.e. they cannot spread the infection to other people) but people with a TB disease can easily transmit the causative agent of TB to susceptible non-infected individuals.

LABORATORY DIAGNOSIS OF MYCOBACTERIUM TUBERCULOSIS INFECTION

The primary specimen for the laboratory diagnosis of a potential TB disease is sputum that is collected in a screw-cap, leak-proof sample container. The reason for using a screw-cap, leak-proof sample container in collecting sputum instead of the usual snap-closing containers is to minimize the spread of the disease or pathogen through aerosols which non-screw-capped, leak-proof sample containers are capable of initiating. Blood, laryngeal swab, bronchoscopic specimens, pleural and peritoneal fluids, gastric lavage and CSF can also be collected from infected patients and analyzed for other TB types. Culturing and microscopic technique are usually the two main methods used for the detection of M. tuberculosis from sputum. Mantoux (tuberculin) skin test, DNA probe testing kits for TB, PCR and chest X-ray are other diagnostic tools or measures used for the clinical diagnosis of the disease.

Growth of M. tuberculosis on Löwenstein–Jensen (LJ) medium. Notice the colorless rough surface, which are typical morphologic characteristics of M. tuberculosis growth on LJ medium

However, the isolation of the acid-fast bacterium in culture and a positive microscopic staining technique are often the two most reliable methods for detecting the pathogen. Culturing of sputum specimen for the detection of M. tuberculosis is usually performed in Reference Tuberculosis Laboratories due to the expensive nature of this procedure using selective media such as the Löwenstein-Jensen (Middlebrook) culture medium. M. tuberculosis should be cultured for identification purposes and antimicrobial susceptibility testing. Selective media such as the Löwenstein-Jensen medium is used for the isolation of the bacterium. Because the tubercle bacteria are slow growing, culture medium are normally incubated for weeks and at a temperature range of 35-37oC. In summary, the diagnosis of tuberculosis requires the detection of acid-fast bacilli (AFB) in sputum of infected patients through the Ziehl-Neelsen stain or other reliable staining techniques as previously stated. Then this must be followed by culturing in a selective media for the identification of the pathogen and consequently to determine their susceptibility profiles.

Microscopical examination of a Ziehl-Neelsen acid-fast staining of M. tuberculosis. M. tuberculosis appears red or pink under the microscope as seen in this image. The acid fast stain usually depends on the ability of mycobacteria including M. tuberculosis to retain the colour of the dye (e.g. Ziehl-Neelsen) when treated with mineral acid or an acid-alcohol solution such as Ziehl-Neelsen or the Kinyoun stains.

MANTOUX (TUBERCULIN) TEST

Mantoux (Tuberculin) Test is used as a laboratory diagnostic aid to detect reactions of individuals to tuberculin i.e. purified protein derivative (PPD). A tuberculin is any preparation that contains tuberculoprotein – which is usually obtained by the filtration of a culture of tubercle bacilli. PPD is the test antigen in the mantoux skin test. It is obtained by boiling an old broth culture of M. tuberculosis. The filtrate that results from this boiling (known as old tuberculin) is treated with an acid (e.g. trichloroacetic acid) to precipitate tuberculoprotein. The tuberculoprotein is further washed and prepared into standard aqueous solutions known as PPD. In order words, PPD and tuberculin are used interchangeable and may mean the same thing.

Principle: Mantoux test is a skin test for TB in which PPD is injected intradermally on the forearm of the infected TB patient. It measures host’s delayed-type hypersensitivity (DTH) to PPD or tuberculin. The principle of the Mantoux test is based on the development of a cell-mediated immunity (CMI) and a type IV hypersensitivity reaction at the site of injection. Positive Mantoux (tuberculin) skin test does not necessarily indicate active TB disease instead it only shows exposure to the pathogen. Individuals who have been previously immunized against the disease may also respond positively to the skin test but those who have had no contact with Mycobacterium shows no reaction to PPD i.e. they react negatively to the Mantoux test. Only the isolation and identification of tubercle bacilli provides the proof of a TB disease.

Illustration of reaction seen on the skin when mantoux test is carried out. The circular shape seen at the site of injection represents a positive result; and it shows the host’s delayed-type hypersensitivity (DTH) to PPD or tuberculin.

A positive Mantoux test result does not in any way show the activity of the infection but only indicates that the individual may have been previously infected or vaccinated against the disease in the past. However, prior exposure or infection with other Mycobacteria species and even previous vaccination with the Bacillus Calmette-Guerin (BCG), an attenuated bovine vaccine could result in a false positive test result. Such false positive results should only be deemed suspicious and further analyzed only in cases or places where the BCG vaccine has not been previously used. The clinical value of the mantoux skin test result (whether negative or positive) depends on the prevalence of reactivity in population and on the incidence of primary TB infection in different age groups. For example, a positive mantoux skin test in young adults, children or infants should raise suspicion of the disease. Also, a negative skin test result especially in regions where the disease has been contained and is uncommon is used as an epidemiological tool to rule out the possibility of a TB disease in the population.

TREATMENT OF MYCOBACTERIUM TUBERCULOSIS INFECTION

The successful treatment of TB disease is usually undertaken using multiple drugs to which the tubercle bacilli are susceptible to due to the possibility of the Mycobacteria in developing resistance to a single anti-tuberculosis drug. Thus, single drug regimens are often ruled out when considering therapy for a TB disease patient. Isoniazid (INH) and rifampin are the two drugs of choice used as first-line TB drugs for the treatment and management of a TB disease. Other first-line TB drugs are ethambutol, pyrazinamide and streptomycin (an injectable TB drug).

The second-line TB drugs include kanamycin (injectable), ofloxacin, capreomycin (injectable), amikacin (injectable), ethionamide, ciprofloxacin and cycloserine. Initial TB therapy is usually started with about three – four TB drugs that include the 2 first-line drugs (INH and rifampin) especially when susceptibility studies are still underway. But when susceptibility results become available, a two-drug therapy is given to the patient except in cases when resistance is anticipated, then the drugs can increase to three or four. The course of drug therapy for TB disease usually spans a period of 9 months in which INH and rifampin are administered concomitantly on a daily basis for about 2 months. TB therapy normally takes a longer period to complete because the pathogen is a slow growing organism, and thus longer time is required to kill them.

The use of two drugs in TB treatment helps to prevent the emergence of tubercle bacilli resistant to each of the drug. Second-line TB drugs are used and included in TB therapy when there is toxicity or resistance associated with any of the first-line drugs. Treatment with multiple anti-TB drugs helps to eradicate the tubercle bacilli and prevent the emergence of resistant strains or spread of the infection to non-TB individuals. In most cases, a direct observed therapy (DOT) in which the patients visits the health center or clinic to take their medication. DOT is anti-tuberculosis program where TB patients are regularly monitored to ensure that they take the full course of their medication so that resistance does not develop and the public health is protected. But when patients fail to comply with their TB regimen, the infection becomes reactivated and the individual becomes infectious again.

PREVENTION AND CONTROL OF MYCOBACTERIUM TUBERCULOSIS INFECTION

People living in high risk regions should be vaccinated with the Bacilli Calmette-Guerin (BCG) vaccine. It should also be administered to infants and children so as to protect them from the infection. BCG is a live attenuated bovine vaccine derived from M. bovis; and the name “BCG” was from the two French scientists (Calmette and Guerin) that developed the vaccine in the early 1920’s.

CLOSTRIDIUM BOTULINUM

Clostridium botulinum is a Gram-positive, strict-anaerobic, motile, pleomorphic, catalase-negative, endospore-forming bacillus (rod) that is ubiquitously found in the soil. The endopores of C. botulinum are sub-terminally placed or located on the bacterium and they are oval in shape. C. botulinum is found in the genus Clostridium and class Clostridia; and members are known to cause food spoilage, gas gangrene, botulism and tetanus. Clostridium species are able to ferment a wide variety of organic compounds, and they produce butyric acid, acetic acid, butanol and acetone, and large amounts of gas (CO2 and H2) as end products during the fermentation of sugars. The ability of Clostridium species to produce gas under anaerobic conditions is the reason why most canned foods contaminated by the pathogen are swollen. It is the causative agent of botulism, a non-communicable disease and a type of food poisoning caused by the exotoxin produced by C. botulinum.

Microscopical illustration of the Gram-positive, rod-shaped and spore-forming morphology of C. botulinum. Notice the terminally-placed spore of the organism.

PATHOGENESIS OF CLOSTRIDIUM BOTULINUM INFECTION

Food-borne botulism or C. botulinum infection in humans occurs following the ingestion of food containing preformed exotoxins (neurotoxin) formed by the pathogen. When home-made canned foods are not properly or well heated to kill the contaminating endopores of C. botulinum, food-intoxication is bound to occur in the individual. Most foods that are eaten almost raw i.e. without cooking are mainly the once with contaminating endospores of C. botulinum. Once these foods become contaminated by the pathogen, C. botulinum grows anaerobically to produce its exotoxin. C. botulinum produces seven types of exotoxins (A – G), but human infections are basically caused by the toxigenic types: A, B, E and F. The environment (soil) is the main source of infection with C. botulinum. After ingestion of the exotoxins, the toxin is absorbed by the gastrointestinal tract (GIT) from where it is transported to the peripheral nervous system. Notably, C. botulinum neurotoxin blocks neuroexocytosis of vesicles containing acetylcholine (a neurotransmitter). This phenomenon goes on to prevent nervous stimulation of the host’s muscles that leads to a flaccid paralysis that resembles that caused by tetanus infection but without muscle contractions. Spasm is used for tetanus infection while paralysis is used to describe botulism.

LABORATORY DIAGNOSIS OF CLOSTRIDIUM BOTULINUM INFECTION

The laboratory diagnosis of C. botulinum food-intoxication (botulism) is by the identification of the pathogen in contaminated food samples, intestinal contents and detection of the organism’s toxin in blood (serum) samples of infected individuals. Stool, vomitus and tissue samples can also be obtained from the patients depending on the type of botulism being investigated. Serum is irrelevant in detecting infant botulism in neonates. The causative agent of botulism is not usually cultured but suspect food samples containing C. botulinum can be cultured in specialized selective media such as the Robertson’s cooked meat medium (RCMM) and the lactose egg yolk milk agar under anaerobic conditions at 35oC for about 2-5 days. Identification of the spores of C. botulinum in a stained preparation can also be employed in the presumptive detection of the pathogen.

TREATMENT OF CLOSTRIDIUM BOTULINUM INFECTION

Supportive care is mainly needed to manage food intoxication caused by C. botulinum. However, a polyvalent antitoxin (of equine origin) is administered intravenously with carefulness. But this antitoxin/antiserum is rarely used in some clinical conditions due to the allergic reactions it sparks in patients (especially infants). Respiratory failures (or pharyngeal paralysis) can cause mortality thus proper ventilation should be maintained in all cases using an artificial breathing apparatus to supply oxygen. On the contrary, most cases of infant botulism are self limiting, and neonates recover from the infection when given proper supportive care. Antibiotics (e.g. amoxicillin) can be administered in cases of wound botulism.

PREVENTION AND CONTROL OF CLOSTRIDIUM BOTULINUM INFECTION

Though food intoxication caused by C. botulinum is an uncommon disease, the prevention of botulism is important and lies in the proper cooking of our foods. Since adequate pressure cooking and autoclaving kills the endopores of C. botulinum, these measures must always be applied in the processing of our foods.

ENTEROBACTERIACEAE

Enterobacteriaceae family contains a large number of bacterial genera that are biochemically and genetically related to one another. Bacteria in this family include: Escherichia, Shigella, Salmonella, Enterobacter, Proteus, and Yersinia.

Common characteristics of Enterobacteriaceae

Members of the Enterobacteriaceae family posses the following characteristics:

  1. They are Gram negative, short rods
  2. They are non-sporulating, facultative anaerobes
  3. They have simple nutritional requirements. MacConkey (MAC) agar is used to isolate and differentiate organisms of Enterobacteriaceae family. Lactose fermenters (LFs) form pink-red colonies on MAC while non-lactose fermenters (NLFs) form pale colored colonies on MAC. LFs include Citrobacter, Escherichia, Enterobacter, and Klebsiella. NLFs include Shigella, Yersinia, Proteus, Salmonella.
  1. Motility if present is by means of peritrichous (lateral) Shigella and Klebsiella are non-motile.
  2. They are catalase positive
  3. They are Cytochrome C oxidase negative
  4. They reduce nitrate to nitrite
  5. They have antigenic cell wall that aid in their identification in the laboratory. The antigens of Enterobacteriaceae are: O:Outer membrane; H: Flagella, K: Capsule; and Vi antigen: Capsule of Salmonella
  6. They produce acid from glucose

Tests for identification of members of Enterobacteriaceae family

Member of the Enterobacteriaceae family are identified based on their biochemical properties. The ommonly used biochemical tests to identify them are:

  1. Citrate utilization Test
  2. Indole Test
  3. Motility Test
  4. Methyl Red (MR) Test
  5. Voges–Proskauer (VP) Test
  6. Triple Sugar Iron (TSI) Agar Test
  7. Urease Test

ESCHERICHIA COLI

Escherichia coli is a facultative, enteric, Gram-negative, motile/flagellated, and lactose-fermenting rod that occur in the genus Escherichia and family Enterobacteria or Enterobacteriaceae. Enterobacteriaceae are bacteria that naturally exist in the intestinal tract of animals and humans, and also found in water and soil. Because the natural habitat of E. coli is the intestinal tract of humans (animals inclusive), it is therefore used as an indicator of the feacal contamination of drinking water and water used for other domestic and industrial purposes. As a baseline, 100 ml of drinking water must not contain any trace of E. coli. While most enteric Gram-negative bacteria such as Shigella and Salmonella are important and regular human pathogens, E. coli are members of the normal intestinal flora and may only cause disease by chance.

Illustration of the Gram negative staining of E. coli. Under the microscope, E. coli appears as a rod-shaped organism with a pink or red colour.

PATHOGENESIS OF ESCHERICHIA COLI INFECTION

E. coli is the most common normal flora of the intestinal tract of humans, and they can also be found in the genital tract and upper respiratory tracts in traces. The presence of pathogenicity islands (acquired foreign DNA) in the genome of enteropathogenic E. coli enhances the pathogenicity and/or virulence of the organism. Infections resulting from E. coli occur occasionally and this has been attributable to the relocation of the bacteria from its normal location in the intestine to other extra-intestinal sites. E. coli causes UTI, diarrheal diseases, meningitis, wound infections and peritonitis. They produce various types of toxins including shiga toxin, labile toxin, stable toxin amongst others; and these help to increase their virulence in the host cells.

Though E. coli is implicated in a handful of human infections when they have the opportunity, diarrheal diseases and UTIs are amongst the two most important infections that characterize the majority of hospital visits across the world. Urinary tract infections caused by E. coli usually arise from the entry of uropathogens into the bladder. Uropathogens can gain entry into the human bladder through sexual intercourse or some minor strain experienced during sexual activity. Urinary tract infections (UTIs) are caused by the presence and growth of bacteria anywhere in the urinary tract system including the kidney, bladder and the urethra. Most cases of UTIs are asymptomatic but some are symptomatic and may present with some clinical signs and symptoms such as increased frequency of urination, dysuria and haematuria (i.e. blood in urine). UTIs affect either the upper urinary tract system (kidney and ureter) or the lower urinary tract system (the bladder and the urethra) of both males and females.

PATHOGENIC STRAINS OF ESCHERICHIA COLI THAT CAUSES DIARRHEAL DISEASES IN MAN

1. ENTEROPATHOGENIC E. COLI (EPEC): EPEC strains causes diarrhea in infants and children in developing countries. EPEC strains adhere strictly to the epithelial cells of the intestines by means of an adhesion molecule and start proliferating. They cause lesions known as effacing or attaching-effacing lesions that affects the microvilli of the intestines, and this lead to profuse and prolonged diarrhea in infants. Vomiting can also be accompanied in an EPEC strain infection. The diarrhea caused by EPEC strains is generally self-limiting but can be chronic and last longer in the infected children. The mode of transmission of this type of E. coli­-associated diarrheal disease is via the feacal-oral route.

2. ENTEROTOXIGENIC E. COLI (ETEC): ETEC strains are the main causative agents of traveler’s diarrhea in people visiting developing countries. It causes watery diarrhea in both infants and adults. ETEC strains produce a variety of enterotoxins that are responsible for the watery diarrhea they produce in their host. The enterotoxins (which are heat-stable and heat-labile in nature) bind to the epithelial cells of the intestines where they stimulate guanylate cyclase that activates the production of cyclic guanosine monophosphate (cGMP). This action mediates the inhibition of sodium ions (Na+) and stimulates the secretion of chloride ions (Cl) and/or electrolytes and water into the lumen of the small intestine that finally result into watery diarrhea. The mode of transmission of this type of E. coli­-associated diarrheal disease is via the feacal- oral route especially through the consumption of foods contaminated with human feaces.

3. ENTEROHAEMORRHAGIC E. COLI (EHEC): EHEC strains are the causative agents of haemorrhagic colitis (a life-threatening bloody diarrhea), and they mainly affect the colon (large intestine) unlike other pathogenic strains of E. coli that attack solely the small intestines. They cause severe abdominal pain followed by bloody diarrhea and haemolytic uraemic syndrome (HUS) in humans. EHEC strains produce verotoxins (a shiga-like toxin) and are sometimes called verocytotoxin-producing E. coli (VTEC) because their toxin is cytotoxic on Vero cells (that originated from kidney cells of African green monkeys) in tissue cultures. Consumption of food stuffs such as beef and other meat products from animals colonized by the bacteria are the main source of acquiring the pathogen. Spinach and unpasteurized fruit juices can also aid in the transmission of EHEC strains. EHEC disease (bloody diarrhea) is a disease of the developed countries. It occurs at a low frequency in developing countries. E. coli 0157:H7 serotype is a major form of EHEC strains as both are genetically related. The mode of transmission of this type of E. coli­-associated diarrheal disease is via the feacal-oral route.

4. ENTEROINVASIVE E. COLI (EIEC): EIEC strains cause E. coli-associated dysentery that resembles shigellosis. Shigellosis is caused by Shigella dysenteriae. Though EIEC infection may occur worldwide, children under the age of 5 years old and who live in developing countries are mostly affected by the disease. EIEC is toxigenic (i.e. it produces enterotoxins), and they penetrate the colonic mucosa and/or epithelial cells of the intestine where they cause inflammatory ulcerations that result in dysentery. The stool of infected patients is usually accompanied with blood, mucous, and pus cells. Contaminated food and water are the main source of acquiring the EIEC infections. The mode of transmission of this type of E. coli­-associated diarrheal disease is via the feacal-oral route.

5. ENTEROAGGREGATIVE E. COLI (EAEC): EAEC strains are the causative agents of food-borne disease in developed countries and chronic and acute diarrhea in people living in developing countries. They cause vomiting and watery diarrhea in infants and children living in developing nations. EAEC strains are notorious in adhering tightly to the intestinal mucosa of its human host to form biofilms or aggregates of bacterial cells. This type of E. coli-associated diarrhea is often associated with neonatal-nursery outbreaks in hospital settings. The mode of transmission of this type of E. coli­-associated diarrheal disease is via the feacal-oral route.

LABORATORY DIAGNOSIS OF ESCHERICHIA COLI INFECTION

The laboratory diagnoses of E. coli-associated diarrheal diseases are mainly based on microscopy and isolation/culture of the infecting pathogen from clinically important specimens. Blood, urine, stool, and pus are some of the specimens obtained for laboratory analysis. A mid-stream urine (MSU) is required to diagnose UTI caused by E. coli in the laboratory. Bacterial counts of E. coli less than 103 colony forming unit (CFU) per ml of urine indicate contamination while counts above 105 CFU/ml of urine are a strong indication of E. coli infection (i.e. significant bacteriuria). E. coli grow on MacConkey agar to produce smooth pink colonies, blood agar (to produce mucoid and haemolytic colonies for some strains), and on cystein lactose electrolyte deficient (CLED) medium (to produce yellow colonies). Molecular detection techniques for prompt identification of E. coli strains include the use of PCR and DNA probes.

E. coli growing on blood agar.

TREATMENT OF ESCHERICHIA COLI INFECTION

Most cases of gastroenteritis caused by bacteria in the Enterobacteriaceae family (E. coli in particular) are self-limiting and often heal without any antibacterial therapy. Since E. coli-associated diarrheal diseases are often accompanied with the loss of fluids and electrolytes from the body, treatment and management of the disease should be started with fluid and electrolyte replacement. Administration of oral-rehydration therapy (containing specific amount of salt, sugar and water) to counter the possible dehydration in the diarrhea patient is the best form of supportive therapy because it returns the affected individual to a normal fluid and electrolyte of the body.

PREVENTION AND CONTROL OF ESCHERICHIA COLI INFECTION

The presence of E. coli in drinking water and water meant for other domestic or industrial purposes is enough indication of feacal contamination of the water source from either sewage or human feaces (especially in places where people defecate in water). Food meant for human consumption becomes contaminated with the pathogen when such water sources are used for food processing. Thus, the prevention of E. coli-associated diarrheal diseases involves avoiding the consumption of contaminated water and food. Travelers or tourists visiting tropical countries should ensure that they only eat properly cooked food and drink only bottled and well disinfected water.

KLEBSIELLA PNEUMONIAE

Klebsiella pneumoniae is a Gram-negative, encapsulated, lactose-fermenting, non-motile, facultative rod in the genus Klebsiella and family Enterobacteriaceae. In addition to O and H antigens, K. pneumoniae possess K antigens (that consists mainly of polysaccharides). K antigens are capsular antigens found mostly amongst members of the Enterobacteriaceae family; and in the case of K. pneumoniae, the K antigens are usually external to the somatic antigens (O and H) of the pathogen. K. pneumoniae is a less common pathogen of the intestinal tract of humans and animals than E. coli. K. pneumoniae is notorious in causing bloodstream infections and severe pneumonia in humans. However, Klebsiella species are most commonly found in the soil and water, and some are nitrogen (N2)-fixing bacteria. K. pneumoniae is a well-studied N2-fixing bacterium that is free-living and exists as a chemoorganotroph in the soil. Generally, K. pneumoniae is mostly implicated as the main causative agent of nosocomial- and community-acquired pneumonia and some UTI.       

PATHOGENESIS OF KLEBSIELLA PNEUMONIAE INFECTION

K. pneumoniae causes opportunistic infections in humans. Persons with history of type I diabetes (i.e. diabetes mellitus), alcoholism, respiratory infections and malnutrition are most often affected. The elderly and individuals with weakened immunity are also prone to K. pneumoniae infections. The human colon and respiratory tract are the main reservoir of K. pneumoniae in the body. Some healthy people harbour K. pneumoniae in their respiratory tract and may be prone to a pneumonia anytime their immunity becomes compromised or is weakened. The pathogenesis of K. pneumoniae is enhanced by its large polysaccharide capsules and its ability to produce toxins and other extracellular factors as is typical to members of the Enterobacteriaceae family.

The capsule produced by K. pneumoniae obstructs phagocytosis in the host while the endotoxin causes inflammation, fever and septic shock. K. pneumoniae reaches the lungs where it produces pneumonia (different from that caused by Streptococcus pneumoniae) via inhalation of respiratory droplets from the upper respiratory tract of individuals harbouring the pathogen. A thick, bloody sputum known as currant-jelly sputum is the main clinical symptom presented in individuals whose pneumonia is caused by K. pneumoniae. Aside pneumonia and UTI, bacteraemia and sepsis are other clinical episodes associated with an infection with K. pneumoniae. Feacal contamination of catheters can result to a UTI, an invasion of IV lines and/or bowel defects in individuals with weakened immune system may result in sepsis.

LABORATORY DIAGNOSIS OF KLEBSIELLA PNEUMONIAE INFECTION

Microscopy, isolation and identification of the pathogen is often the mainstay of diagnosing any infection in which K. pneumoniae is implicated or suspected. K. pneumoniae is Gram-negative and non-motile. Characteristically, K. pneumoniae produces mucoid colonies on growth media. K. pneumoniae grows on differential media including MacConkey agar producing mucoid colonies and CLED (producing yellow-mucoid colonies). On blood agar, K. pneumoniae produces large grey or pale mucoid colonies. Biochemically, K. pneumoniae is urease positive, lactose-fermenting and citrate positive. It is also positive to malonate utilization test and lysine decarboxylase (LDC) test. Some species of Klebsiella (e.g. K. oxytoca) is indole positive.

Illustration of the mucoid and slimy nature of Klebsiella on growth media

TREATMENT OF KLEBSIELLA PNEUMONIAE INFECTION

The treatment of K. pneumoniae infection should be guided by a proper susceptibility test result owing to the nature of the pathogen to produce extracellular substances including beta-lactamases that render antimicrobial agents directed towards it ineffective. Some strains of Klebsiella especially those associated with hospital-acquired infections are multidrug resistant; and thus, the treatment of K. pneumoniae infection should be embarked using multiple antibiotic regimens. Third-generation cephalosporins and aminoglycosides are often used for the treatment of both nosocomial and community-acquired K. pneumoniae infections.

PREVENTION AND CONTROL OF KLEBSIELLA PNEUMONIAE INFECTION

The control of K. pneumoniae infection in the community and around hospital settings depends on ensuring proper infection control strategies when handling patients and using invasive and non-invasive medical devices (e.g. catheters, respirators and IV lines). These medical devices known to spread the pathogen should be used cautiously and only when needed.

CLOSTRIDIUM TETANI

Clostridium tetani is a Gram-positive, motile, anaerobic, spore-forming, rod-shaped bacterium found in the genus Clostridium. It is the main causative agent of tetanus or lock jaw (a non-infectious disease) in humans and animals. Tetanus is an acute localized non-communicable disease of man and animals characterized by muscle spasm and caused by spores of C. tetani that enters the body from the soil via a wound site. C. tetani is ubiquitous in the soil; and can also be found in the hospital environments, feacal matter and intestinal tracts of some animals. The skin especially a bruised or wounded one is the main portal of entry of C. tetani into the human body. Human infection begins when spores of C. tetani in the soil enters a wound.

The endospore of C. tetani grows and starts producing exotoxins known as tetanospasmin at the site of infection when the wound site is anaerobic and receives low blood or oxygen supply. Tetanospasmin is a plasmid-encoded neurotoxin produced by C. tetani; and it is the main virulence factor of C. tetani infection. Tetanus toxin (tetanospasmin) has a molecular weight of 150,000 (or 150 kDa) that usually comprises two peptide subunits: fragment A known as light chain (50,000 MW or 50 kDa) and fragment B known as heavy chain (100,000 MW or 100 kDa); and both are joined together by a disulphide bond. Normally, the tetanospasmin is cleaved extracellularly by a bacterial protease into fragments A and B.

PATHOGENESIS OF CLOSTRIDIUM TETANI INFECTION

Endospores of C. tetani are introduced into the body through burns, surgical sutures, wounds or invasive injury on the skin; and thus C. tetani only infect humans by chance via wound or injury on the skin. C. tetani is usually a non-invasive pathogen and only cause disease (tetanus) through the action of its exotoxin (tetanospasmin). Tetanospasmin is produced when C. tetani endospores and vegetative cells germinate under the anaerobic conditions created in the wound. C. tetani only multiplies locally at the wound site without spreading or causing any significant damage to neighboring tissues but as the injury site becomes anoxic, it grows to produce the tetanus toxin (tetanospasmin) which is carried to the central nervous system (CNS) where it impacts the spinal cord and brain to cause series of unrestrained muscular stimulations.

Tetanospasmin (tetanus toxin) binds to gangliosides in synaptic membranes of CNS where it inhibits and suppress the release of inhibitory neurotransmitters (e.g. glycine and gamma-amino butyric acid, GABA), thus causing muscle spasm in key muscular/skeletal sites of the affected individual’s body. The blockage of the release of inhibitory neurotransmitters such as glycine and gamma-amino butyric acid (GABA) leads to convulsive contractions of voluntary muscles in the affected human or animal host. Generally, this leads to difficulty in swallowing, increased muscle tone, lock jaw/trismus (difficulty in opening the mouth) and rigidity of the muscles. The incubation period of tetanus is variable and often shorter spanning days or weeks after the initial introduction of C. tetani spores into wound sites on the body. Clinical symptoms of tetanus may include trismus or lockjaw (i.e. inability to open the mouth properly), and this may later progress to spasm or muscular contractions in other sites of the body such as the pharyngeal muscles (which affects swallowing).

Risus sardonicus (i.e. spasm of the facial muscles) and opisthotonos (i.e. backward contraction of the back towards the heels) are other complications of the disease. Spasm of the respiratory muscles results in respiratory failures; and this may cause death. Tetanus is a disease with a high mortality rate. In some parts of the world such as Asia and Africa, neonatal tetanus can ensue from failure of observing aseptic technique during childbirth such as when contaminated instruments are used to cut the umbilical cord of the foetus after delivery. Neonatal tetanus can also occur from improperly or non-immune mothers; and in such situation, the child has not acquired passive immunity from the mother and the umbilical cord can become infected in the process of birth. Female circumcision, unskilled abortion and performing surgery with contaminated or unsterile instruments are other predisposing factor that may cause tetanus in some developing countries.           

LABORATORY DIAGNOSIS OF CLOSTRIDIUM TETANI INFECTION

There is rarely any routine laboratory test for the diagnosis of tetanus. However, the clinical diagnosis of the disease in most cases lies on the history of the wound infection and clinical symptoms of the disease (e.g. muscle stiffness) in the individual. Gram staining shows the presence of bacilli with terminal spores that resembles a tennis racket. Examination of blood or tissue for the presence of tetanospasmin is rarely performed but culture can be carried out on selective media to isolate the organism. C. tetani is indole positive.

TREATMENT OF CLOSTRIDIUM TETANI INFECTION

Treatment of tetanus should be perfumed without delay using anticonvulsive drugs and regimens that prevent spasm. Tetanus toxin should be neutralized in the affected individual via the administration of antitoxin such as the human tetanus immune globulin, HTIG (human antitoxin). Wound infections especially those contaminated with soil should be properly taken care of by dressing and the administration of antibiotics such as penicillin which inhibits the growth of C. tetani and toxin production. Sedatives such as benzodiazepines should be given to patients already experiencing muscle spasm; and they should be placed in a quiet dark location and given respiratory support to maintain sufficient airflow in the airway system.

PREVENTION AND CONTROL

Tetanus is a preventable disease. It can be prevented through the use of immunization or vaccination of susceptible human population using tetanus toxoid vaccine. Massive and sustained active immunization against tetanus (though short-lived) is necessary in preventing tetanus in children of developing countries where the mortality rate of the disease is high.

STAPHYLOCOCCUS AUREUS

Staphylococcus aureus is a Gram-positive, coagulase-positive, catalase-positive, non-motile coccus found in the genus Staphylococcus and family Staphylococcaceae. They are facultative anaerobic organisms, and they cause haemolysis on blood agar. Staphylococcus species are usually arranged in groups, in pairs, tetrads and they also occur singly. S. aureus usually appear as grapelike clusters under the microscope. They are asporogenous or non-sporulating in nature. Asporogenous bacteria are organisms that do not produce spores. S. aureus are habitually found in the nose of humans but it may be found regularly in most other anatomical sites of the body such as the respiratory tract, mucous membranes, GIT and skin. S. aureus is mostly implicated in human pyogenic infections such as boils, pimples, impetigo and pustules.

S. aureus appears as a coccus, with a purple colour under the microscope.

PATHOGENESIS OF STAPHYLOCOCCUS AUREUS INFECTION

S. aureus is notorious in causing a variety of invasive and mixed infections in humans including pus-forming infections, food-poisoning (gastroenteritis) characterized by vomiting, skin infections and blood-borne related diseases. Urinary tract infections (UTIs), pneumonia, mastitis, endocarditis, meningitis and osteomyelitis are some of the serious infections or diseases in which S. aureus is implicated as a causative agent. The pathogenesis of S. aureus is based on the virulence factors (i.e. enzymes and toxins) that they produce. The toxins produced by S. aureus are:

  • EXFOLIATIN: Exfoliatin or exfoliative toxins (ET) are protein toxins produced by Staphylococcus aureus strains that cause staphylococcal scalded skin syndrome (SSSS) in humans.
  • TOXIC SHOCK SYNDROME TOXIN-1 (TSST-1): Toxic shock syndrome toxin-1 (TSST-1) is another class of superantigens produced by aureus, and it causes toxic shock syndrome disease in humans.
  • ENTEROTOXINS: Enterotoxins are superantigens produced by pathogenic aureus strains that cause toxicoses, a type of food poisoning in humans.
  • STAPHYLOCOCCAL ALPHA (α) TOXIN: Staphylococcal alpha (α) toxins are cytolytic toxins produced by pathogenic aureus, and which has killing effect on the cell membranes of eukaryotic cells.
  • STAPHYLOCOCCAL BETA (β) TOXIN: Staphylococcal beta (β) toxins are less cytotoxic than α toxin but they also attack erythrocytic cells and some cells of the nerves (e.g. sphingomyelin). Beta toxins have high affinity for lipid-rich cells where they cause haemolysis.
  • STAPHYLOCOCCAL GAMMA (γ) TOXIN: Staphylococcal gamma (γ) toxins are produced by both aureus and S. epidermidis. Gamma toxins disrupt the integrity of cell membranes like the other haemolysins produced by S. aureus.
  • LEUKOCIDIN: Leukocidin is a cell membrane damaging toxin produced by pathogenic strains of aureus. They specifically kill leukocytes by creating small pores or holes that increases loss of materials from the damaged cell, thus inhibiting the process of phagocytosis in the infected human host.

EXTRACELLULAR ENZYMES PRODUCED BY S. AUREUS

  • Catalase: Catalase is an enzyme produced by pathogenic aureus, and it inhibits the process of phagocytosis. It enhances the survival of the pathogen in phagocytes through the production of the enzyme. Catalase enzyme production is used for the biochemical identification of S. aureus in the laboratory, and it converts hydrogen peroxide (H2O2) to water and oxygen; thereby walling off or protecting the infected body sites from phagocytic cells.
  • Proteases: Proteases or proteinases are extracellular enzymes produced by pathogenic aureus, and which assist the pathogen in breaking down protein molecules.
  • Nuclease: Nuclease enzymes produced by pathogenic aureus breaks down nucleic acids of infected cells in human host. DNase enzymes produced by pathogenic S. aureus perform a similar function with nucleases in that they destroy the host cell DNA.
  • Beta-lactamase: Beta lactamase enzymes produced by pathogenic aureus are of clinical significance in that this class of enzymes confers on the pathogen the capacity to develop resistance to a range of synthetic antibiotics and other antimicrobials. Beta lactamase enzymes of pathogenic S. aureus degrade beta-lactam antibiotics such as penicillins.
  • Lipase: Lipases are fat or lipid destroying enzymes produced by pathogenic aureus.
  • Coagulase: Pathogenic aureus produces coagulase enzymes, and this characteristic differentiates them from non-pathogenic S. aureus strains which do not produce coagulase. The production of this enzyme by pathogenic S. aureus strains inhibits the process of phagocytosis in the affected human host cells. Coagulase enzymes converts fibrinogen to fibrin clot which surrounds and protect infected sites from the action of phagocytes. The production of coagulase (blood clotting factor) is used to identify pathogenic S. aureus in the clinical microbiology laboratory.
  • Staphylokinase: Staphylokinase is an extracellular enzyme produced by pathogenic aureus, and it is a plasminogen activator (i.e. the enzyme stimulates a plasmin-like proteolytic activity that lyses fibrin). Streptokinase may aid in the spreading of the pathogen within the host due to its ability to degrade fibrin clots.
  • Hyaluronidase: Hyaluronidase is an enzyme that breaks down hyaluronic acid that makes up the host connective tissues. The ability of pathogenic aureus to produce hyaluronidase encourages the spread of the pathogen in host tissues.
  • Protein A: Protein A is found in the cell wall of most aureus strains, and they prevent the activation of complement in the host cell. Anti-phagocytic in nature, protein A binds to the crystallizable fragment (Fc portion) of antibody molecules (e.g. IgG), and thus prevents phagocytosis and opsonization. In this way, protein A (staphylococcal surface protein) contributes to the virulence of pathogenic S. aureus.

LABORATORY DIAGNOSIS OF STAPHYLOCOCCUS AUREUS INFECTION

The laboratory diagnosis of staphylococcal disease is based mainly on the isolation and identification of the invading pathogen through microscopy and culture. Serological and biochemical tests (e.g. catalase, DNase and coagulase tests) are also employed in typing the strain of S. aureus implicated in the disease process. Blood, CSF, sputum, tracheal aspirate, pus, and surface swab specimens from infected sites (e.g. wound and burns) are clinical specimens collected for laboratory investigations. Gram staining reveals Gram-positive grape-like cocci in clusters, tetrads or pairs under the microscope.

S. aureus produces grape-like clonies that can either be in clusters or in tetrads on blood agar.

Mannitol salt agar (MSA) is a selective medium (that contains NaCl which inhibit other normal flora and non-staphylococcal organisms) used to screen for S. aureus and recover the pathogen from specimens resulting from a mixed infection. S. aureus produces several types of haemolysis including beta-haemolysis, alpha haemolysis and gamma haemolysis on blood agar media. S. aureus can also be cultured and isolated successfully on blood agar producing white or pale haemolytic colonies, chocolate agar and MacConkey agar. S. aureus grow aerobically at 35-37oC.

TREATMENT OF STAPHYLOCOCCUS AUREUS INFECTION

Therapy for staphylococcal disease is based on the administration of specific class of antibiotics to which the pathogen is susceptible to. All isolated Staphylococcus species should be subjected to antimicrobial susceptibility studies so as to guide treatment. Staphylococcal food poisoning should be treated through fluid and electrolyte replacement by the administration of the correct amount of a salt-sugar-solution (SSS) to the affected patients since the disease normally leads to a considerable loss of fluid from the body.

PREVENTION AND CONTROL OF STAPHYLOCOCCUS AUREUS INFECTION

Staphylococcus species are habitual inhabitants of the human body especially the nares and skin where they are resident as normal microflora. Most individuals who harbour pathogenic S. aureus are asymptomatic, and they shed the pathogen to susceptible hosts around them. The control and prevention of staphylococcal infections in the hospital environment should be based on the practice of proper hospital infection control measures such as hand washing and disinfection.

STREPTOCOCCUS PYOGENES

Streptococcus pyogenes is a Group A Streptococcus species that is non-spore forming, Gram-positive, non-motile round to ovoid bacterium that occur in chains, singly (as coccus) or in pairs (as diplococci). It is the most common cause of pharyngitis (sore throat) in humans. Other streptococcal diseases caused by Streptococcus species in humans include scarlet fever, cellulitis, necrotizing fasciitis, erysipelas, impetigo, tonsillitis, pneumonia, otitis media, and septicaemia. S. pyogenes is a beta-haemolytic Streptococcus species that is catalase negative and facultatively anaerobic. It requires enriched media (e.g. agar supplemented with sheep blood) to grow effectively in the laboratory.

Streptococcus species are complex bacterial species classified into different groups based on their clinical significance and ability to haemolyse or breakdown blood. Some of the notable Streptococcus groups are Group A streptococci (e.g. S. pyogenes) which cause beta-haemolysis, Group B streptococci (e.g. S. agalactiae) which cause α, β, and γ haemolysis, Viridans streptococci group (which contain non-typable or groupable Streptococci species such as S. mutans), Group D streptococci (e.g. Enterococcus faecalis and S. bovis), and other untypable or ungroupable Streptococcus species including S. pneumoniae (pneumococcus) that has no specific antigenic substance for their grouping.

The groupings of Streptococcus species is based on serological data and/or specific antigenic structures associated with each of the streptococcal species, and this classification is based on the Lancefield classification – which classifies bacteria according to specific cell wall and capsular antigens found on the bacteria. E. faecalis is also known as S. faecalis. Lancefield classification (as described by the famous microbiologist Rebecca Lancefield) grouped Streptococcus species into groups A-O. The throat and skin are the main reservoir of S. pyogenes in the human body.     

PATHOGENESIS OF STREPTOCOCCUS PYOGENES INFECTION

S. pyogenes causes infections of the upper respiratory tract and other invasive infections of the skin and connective tissues of humans. Like Staphylococcus species, S. pyogenes produces a wide variety of virulence factors which spur the many diseases that they produce in humans. Some of the extracellular enzymes and toxins produced by S. pyogenes include streptolysin O and S, streptokinase, Dnase, hyaluronidase, M protein, haemolysins, and streptococcal pyrogenic exotoxins. Streptolysin O (SO) is an immunogenic, haemolytic and cytolytic toxin produced by S. pyogenes. It is antigenic in nature, thus stimulating the production of anti-streptolysin O (ASO) antibody in infected individuals; and can be used to diagnose recent S. pyogenes infections. SO is oxygen-labile (i.e. it is not active in the presence of oxygen), and is thus inactivated by oxidation.

Streptolysin S (SS) is a non-immunogenic, haemolytic, oxygen-stable toxin produced by S. pyogenes. SS is not antigenic, and thus forms no antibody in infected individuals. Both SO and SS destroy the cell membranes of red blood cells (RBCs) and other host cell membranes. Streptococcal pyrogenic exotoxins also known as erythrogenic toxin is mainly responsible for the rashes produced in scarlet fever infection; and the toxins (which are superantigens) produces similar clinical signs as that of toxic shock syndrome toxin (TSST) caused by S. aureus. M protein (fibronectin-binding protein) appears as hair-like structures on the cell wall of S. pyogenes; and it is primarily responsible for protecting the pathogen from host phagocytic action. S. pyogenes gains entry into the human body through mucous membranes or fractured skin; and the pathogen multiplies rapidly and evades some mechanisms of the host immune system (e.g. phagocytosis).

Upon entry, the pathogen causes a variety of acute, invasive and systemic infections such as impetigo (infection of the superficial layers of the skin), scarlet fever, a widespread skin rash, rheumatic fever (autoimmune disease involving the joints), necrotizing fasciitis (inflammation of skeletal muscles), pharyngitis (sore throat infection), tonsillitis (inflammation of the tonsils), cellulitis (infection of the deep layers of the skin), puerperal fever (sepsis after childbirth) and other severe invasive infections that may affect the heart and kidney and other vital organs of the body.

LABORATORY DIAGNOSIS OF STREPTOCOCCUS PYOGENES INFECTION

The laboratory diagnosis of S. pyogenes infections is based on the isolation and identification of the pathogen in culture and through microscopic and serological tests. Throat swab, wound swab, blood, pus and swabs from skin lesions are some clinically important specimens required for the laboratory diagnosis of S. pyogenes infections. Latex agglutination tests that detect ASO produced by S. pyogenes are commercially available for laboratory diagnosis of the disease. Gram stained smears of wound infections and skin lesions may prove diagnostic but stained smears of samples in pharyngitis are clinically irrelevant because Viridans Streptococci species are members of the human normal flora and may be indistinguishable from S. pyogenes in stained smears. Rapid diagnostic test kits (e.g. ELISA) are usually employed in the laboratory diagnosis of sore throat caused by S. pyogenes. S. pyogenes grow on blood agar and chocolate agar to produce small haemolytic colonies. S. pyogenes is susceptible to bacitracin but not optochin; and biochemically, it is catalase negative.

S. pyogenes grow on blood agar and chocolate agar to produce haemolytic colonies.

TREATMENT OF STREPTOCOCCUS PYOGENES INFECTION

Therapy for S. pyogenes infections is based on antimicrobial susceptibility test result and the type of prevailing disease. Penicillin G, erythromycin, cephalosporins and fluoroquinolones are some of the antibiotics of choice used to manage infections caused by S. pyogenes. Treatment of Streptococcal infections is important in order to prevent the spread of the pathogen to susceptible individuals and also to ease the already infected patient from the symptoms of the disease.  

PREVENTION AND CONTROL OF STREPTOCOCCUS PYOGENES INFECTION

No vaccine currently exists for immunization against diseases caused by S. pyogenes. Vaccines are only available for infections caused by S. pneumoniae. Prevention and control of S. pyogenes infection transmission is usually based on effective treatment of infected individuals. Infected individuals should be properly treated, and contact with non-infected people should be restricted as much as possible.

STREPTOCOCCUS PNEUMONIAE

Streptococcus pneumoniae is a Gram-positive, capsular, non-motile and catalase-negative cocci that are usually lancet (bullet) shaped. S. pneumoniae also occurs in pairs (diplococci) or in chains (streptococci); and they are haemolytic in nature and inhibited by optochin. A member of the normal flora of the human upper respiratory tract system, S. pneumoniae (commonly referred to as pneumococcus), is notorious in causing a range of infections in human beings. It is the most common cause of community acquired pneumonia in humans. Other infections in which S. pneumoniae is implicated are osteomyelitis, abscesses, otitis media, meningitis, bacteraemia, peritonitis, endocarditis and cellulitis.

Virulent strains of S. pneumoniae have surface capsules composed mainly of high-molecular monosaccharides and oligosaccharides that interfere with phagocytosis. The upper respiratory tract of humans is the main reservoir of pneumococci, and they can be spread to susceptible host via respiratory droplets through this route. Pneumococci colonize the upper respiratory tract without causing any disease, but infection ensues following weakened immune system and poor state of health. Pneumonia caused by S. pneumoniae is most prevalent amongst certain individuals with some predisposing factors such as prior viral infection of the respiratory tract, history of alcoholism, drug intoxication, injury to the respiratory tract, heart failure, diabetes and old age amongst others.

PATHOGENESIS OF STREPTOCOCCUS PNEUMONIAE INFECTION

Pneumonia (reduced function of the lungs) is a lung disease that is caused by certain pathogenic bacteria species including S. pneumoniae (pneumococcus). Other bacteria that cause human pneumonia are Staphylococci, Haemophilus, Pseudomonas, Mycoplasma and Chlamydia. Non-bacterial causes of pneumonia are some species of viruses, fungi and protozoa. The virulence of infection caused by S. pneumoniae is usually determined by some virulent factors such as pili, capsules, cell wall surface proteins (teichoic acid and peptidoglycan), haemolysins, hydrogen peroxides and autolysins. The onset of pneumonia in humans starts following the aspiration of respiratory secretions that contains virulent strains of S. pneumoniae.

Upon invasion and possible colonization of the upper respiratory tract where they normally inhabit, S. pneumoniae reaches the lower respiratory tract where they attack the alveolar cells and produce a variety of lung diseases. S. pneumoniae multiplies rapidly in the alveolar spaces, and produce inflammatory cells which fills the alveoli and/or lungs. Systemic infections due to pneumococcus occur when S. pneumoniae reaches the body’s blood circulation via the lymphatic vessels of the lungs. S. pneumoniae can reach other sites of the body such as the middle ear, heart and meninges via the respiratory tract and produce further clinical complications. Fever, chills, chest pain, difficulty in breathing and expectoration of sputum usually accompanied with blood (i.e. rusty coloured blood) are some of the clinical signs and symptoms associated with pneumonia caused by S. pneumoniae.

Bronchial pneumonia and lobar pneumonia are the two types of pneumonia caused by pneumococcus in humans. Bronchial pneumonia is common in old people, young children and infants while lobar pneumonia is commonly experienced by young adults. Uncapsulated strains of pneumococcus are generally avirulent, and are not capable of producing pneumonia in humans. Only the capsulated S. pneumoniae strains actually produce the clinical episodes of the disease, and this is because capsulated pneumococci have polysaccharide capsules that protect them from phagocytosis.     

LABORATORY DIAGNOSIS OF STREPTOCOCCUS PNEUMONIAE INFECTION

The laboratory diagnosis of pneumonia caused by S. pneumoniae is mainly based on the identification and isolation of the pathogen from patient’s specimens via microscopy and culture techniques. Sputum, CSF, exudates and blood specimens are usually the main samples collected when pneumococcal pneumonia is suspected. Gram stained smear of sputum samples reveals Gram-positive lancet (bullet) shaped diplococci. S. pneumoniae is a fastidious bacterium, and they grow best in culture media supplemented with horse, rabbit or human blood and incubated in 5% carbon dioxide.

On blood agar, S. pneumoniae produce mucoid or translucent colonies that are alpha haemolytic; and pneumococcus is optochin sensitive and soluble to bile salts. Biochemically, pneumococcus ferment glucose to produce lactic acid. Serologically, S. pneumoniae is positive to the quellung reaction test also known as the swelling reaction. In quellung reaction test which is used to determine the polysaccharide capsule of S. pneumoniae, the capsules of pneumococcus swells markedly when the pathogen is mixed with certain specific antiserum and then examined microscopically at 1000X for capsular swelling. Quellung reaction test is usually used for the rapid detection of pneumococcus from cultures and sputum samples in the microbiology laboratory.

TREATMENT OF STREPTOCOCCUS PNEUMONIAE INFECTION

Pneumonia caused by pneumococcus is stopped abruptly when the right antimicrobial agents are administered early. Penicillins V & G, vancomycin, cephalosporins, macrolides and quinolones are some of the drug classes used to treat and manage pneumococcal pneumonia. Some strains of pneumococcus may be resistant to some first-line antibiotics such as penicillins, thus antibiotic therapy should be guided by the results of susceptibility studies.

PREVENTION AND CONTROL OF STREPTOCOCCUS PNEUMONIAE INFECTION

The prevention of pneumococcal pneumonia is largely based on immunization using pneumococcal vaccine and the effective treatment of affected individuals.

SPIROCHAETES

Spirochaetes (which are coiled, helical and actively motile bacteria) normally exists as free-living organisms (or commensals), parasites or important human and animal pathogens. They can be found in mollusks, insects, animals, in the soil and water. Treponema pallidum is the most important human pathogen in the group of microorganisms known as spirochaetes. Leptospira species and Borrelia species are other important spirochaetes that cause significant human infections. Leptospira species causes leptospirosis while Borrelia species cause lyme disease.

TREPONEMA PALLIDUM

Treponema pallidum (well known scientifically as T. pallidum subsp. pallidum) is a Gram variable or Gram-negative, microaerophilic or anaerobic, motile, spiral-shaped bacterium (spirochaete) that is found in the genus Treponema and family Spirochaetaceae. It is the etiologic agent of syphilis, a sexually transmitted disease (STD) in humans. Syphilis is a contagious STD like gonorrhea, and the disease occurs worldwide in both males and females at varying frequency. T. pallidum subsp. pallidum which causes venereal syphilis and congenital syphilis (acquired by the feotus in utero from syphilis infected expectant mothers) is the main important human pathogen in the Treponema genera.

Electron micrograph (EM) of T. pallidium. Notice the spiral-shaped morphology of the organism.

However, other Treponema species that cause significant human infections (e.g. yaws or endemic syphilis caused by T. pallidum subsp. Endemicum) also exist. T. pallidum subsp. pallidum (known as T. pallidum) hardly grow outside the human body. This makes in vitro cultivation of the bacteria in the laboratory impossible, and thus hampers further investigations of its metabolic pathways and other significant characteristics.

PATHOGENESIS OF TREPONEMA PALLIDUM INFECTION

Syphilis infection is strictly a human disease that has no animal linkage. T. pallidum gains entry into the human body mainly by direct body contact when there is a break on the skin or epidermis and especially during sexual intercourse with an infected partner (having lesions containing the organism). Ulcerative lesions (containing Treponema pallidum) on the mouth (particularly in those who engage in oral sex), genital organs (penis, cervix or vagina) and the rectum (homosexuals in particular) are usually the main route of transmission of the pathogenic bacteria to susceptible human host.

Direct body contact with the blood or mucosal surfaces of infected persons can also serve as means by which the pathogen is transmitted to susceptible human hosts. Mother to child transmission in utero (i.e. in congenital syphilis) is also possible. Upon invasion, the pathogen migrates to the lymph nodes after multiplying at the portal of entry and then becomes systemic and reaches the bloodstream where it causes bacteraemic conditions in the infected host. The pathogenesis of syphilis infection is complex and thus can be described in a number of steps. Infected untreated individuals usually experience three types or phases of syphilis infection.

PRIMARY SYPHILIS: Primary syphilis which is also known as stage I syphilis is characterized by a localized lesion which can occur on the mouth or in the genitalia including the cervix, the vagina and the penis. Chancre or sore due to primary syphilis infection as aforementioned can also be observed on the tongue and inside or within the walls of the vagina of infected females. This localized lesion which usually forms in about 2 months after initial infection is known as hard chancre. It is painless, and is usually formed at the point of entry of the pathogen (in either the mouth or genital region) as a sore or boil. Primary syphilis usually heals spontaneously without any formal treatment (except in HIV infected persons) but the pathogen disseminates to other vital organs of the body (e.g. CNS and the eyes) via the bloodstream.

SECONDARY SYPHILIS: Secondary syphilis or stage II syphilis usually occurs 2-3 months after the self healing of the primary syphilis. It is characterized by a dissemination of the pathogen in the body of the infected individual. In stage II syphilis, there is a generalized body rash known as maculopapular rash which appears all over the body of the infected individual. Fever, headache, body pain and malaise are some of the signs and symptoms that are usually associated with this stage of the disease. Secondary syphilis can be asymptomatic with disease progression, and some vital organs of the body such as the liver may be affected. Stage II syphilis like primary syphilis heals spontaneously even without treatment. However, the disease may progress into a latent period during which there are no clinical signs and symptoms of the disease but there is the presence of an infection (which can be proved in the laboratory by serodiagnosis). Latency or latent period of syphilis infection usually last for about 2 years (for early latency) or 4 years (for late latency), and it later reappears in the individual as tertiary syphilis infection if the immune system of the host fails to clear it from the body. Infected individuals are usually infectious at the early latent period but remain noninfectious at the late latent period of secondary syphilis infection.

TERTIARY SYPHILIS: Tertiary syphilis manifests in many organs of the body as generative lesions generally known as granulomas, gummas or focal lesions. Organs of the body affected in stage III syphilis include the skin, meninges, bone, liver, brain, and the heart amongst others. Stage III syphilis is not infectious and it occurs after many years in some individuals who have secondary syphilis infection. Tertiary syphilis is the result of an untreated syphilis infection, and it can be fatal especially when the CNS is affected resulting in severe neurological disorders amongst other lethal disorders (e.g. aortic aneurysm, loss of sight and dementia). Treponemes can rarely be found in stage III syphilis unlike in stage I and state II where the pathogen can be assayed and identified in the specimens of infected persons.

CONGENITAL SYPHILIS: Congenital syphilis is a non-STD type of syphilis that occurs in the unborn child in which T. pallidum is transmitted from an infected mother to the foetus in utero. Loss of pregnancy or abortion, stillbirth, early birth and the death of the infant are some of the fatalities associated with congenital syphilis infection (which is transplacentally-acquired). Newborns with syphilis infection and who may have survived the disease, usually progress in life with signs and symptoms of the infection and other several anomalies associated with T. pallidum infection in neonates (e.g. blindness, paresis, mental retardation, psychosis et cetera).

LABORATORY DIAGNOSIS OF TREPONEMA PALLIDUM INFECTION

The laboratory diagnosis of syphilis infection is usually based on serodiagnosis and parasite demonstration in stained smears. Biopsy specimens and serum from blood are the samples of choice required for analysis. In vitro cultivation of T. pallidum is still impractical due to the inability of the pathogen to grow outside the human body. Though cell/tissue culture can be used in studying the bacteria pathogen, T. pallidum subculture in tissue culture is not viable.

Motile and corkscrew or spiral-shaped treponemes can be demonstrated in darkfield microscopy from genital lesions (excluding lesions at the oral or anal region) and immunoflourescence microscopy. Note: Lesions at the oral or anal region of syphilis infected individuals should not be examined by darkfield microscopy due to the possibility of a false positive result because some Treponema species are normal flora of these body sites. Several serological tests (e.g. VDRL and RPR) are commercially available for the serological diagnosis of syphilis infection in the laboratory.

TREATMENT OF TREPONEMA PALLIDUM INFECTION

The antibiotic of choice for treating syphilis infection (latent, primary and secondary stage inclusive) is penicillin G which is usually administered extensively and via the parenteral route. Other antibiotics used include cephalosporins, erythromycin and tetracyclines; and these are usually used to treat T. pallidum infected individuals who may be allergic or non-responsive to penicillin G.

PREVENTION AND CONTROL OF TREPONEMA PALLIDUM INFECTION

The primary means of acquiring T. pallidum infection is via sex with an infected person. The only exception is congenital syphilis which is transmitted from an infected mother to the unborn child. Thus, the best way of preventing a syphilis infection is by practicing safe sex, avoiding unsafe sexual practices such as anal sex and oral sex, and by treating any primary infection that occurs. Infected individuals should ensure that their sex partners are treated as well so as to avoid re-infection. Congenital syphilis can be prevented in neonates by adequate screening and treatment of infected expectant mothers prior to their child delivery. No vaccine currently exists for preventing syphilis infection.

LISTERIA MONOCYTOGENES

Listeria monocytogenes is a Gram-positive, catalase-positive, non-spore forming, aerobic or anaerobic intracellular rod bacterium in the genus Listeria and family Listeriaceae. L. monocytogenes can also exist as coccobacilli or short chains (resembling Streptococcus and Corynebacteria species) and as saprophytes in the environment. Its motility varies with temperature. L. monocytogenes is motile at 18-20oC with a tumbling motility in liquid media but at 37oC, the bacterium is weakly motile or non-motile. L. monocytogenes is the main causative agent of listeriosis, a food borne illness (gastroenteritis) in humans especially the immunocompromised, the elderly, transplant patients, infants, pregnant women and those taking immune suppressive drugs.

Listeria monocytogenes is a Gram positive bacterium; and thus the organsim appears purple under the microscope after Gram staining.

It causes neonatal sepsis and meningitis in neonates and newborns; and it is also implicated in causing sepsis, meningoencephalitis and bacteraemia in the elderly, pregnant mothers and the immunocompromised who are the most people at risk of an infection with L. monocytogenes. L. monocytogenes is a commonly encountered bacterial pathogen in food products especially vegetables, meat and milk products which they ubiquitously inhabit. The bacterium is a psychrotolerant organism i.e. it can grow at very low temperature such as the refrigerator temperature. Growth at low temperatures allows L. monocytogenes to thrive in refrigerated stored-food products via which likely human infection may occur after consumption.

PATHOGENESIS OF LISTERIA MONOCYTOGENES INFECTION

L. monocytogenes is an environmentally ubiquitous bacterium that commonly contaminates food products including those stored in the refrigerator (e.g. cheese, milk, vegetables and meat). The main route via which the pathogen enters the body is via the consumption of food contaminated with L. monocytogenes. After ingestion, the pathogen moves to the gastrointestinal tract where it adheres and is taken up by macrophages and other non-phagocytic cells (through endocytosis). The attachment and ingestion of L. monocytogenes by phagocytic cells and/or the epithelial cells of the gastrointestinal tract is facilitated by internalins (cell wall surface proteins) produced by the pathogen.

L. monocytogenes is an intracellular bacterium, and it produces listeriolysin O (a membrane liquefying and pore-forming enzyme) within the phagolysosome where it is enclosed. Listeriolysin O (LLO) creates holes on the phagolysosome and promotes rupture, and thus releasing L. monocytogenes into the cytosol of the cytoplasm where further multiplication occurs. Escape of L. monocytogenes from the phagolysosome which is mediated by listeriolysin O prevents damage of the bacterium within the phagosome. This phenomenon allows the bacterium to spread to other nearby cells and even from one epithelial cell to another.

The ability of L. monocytogenes to multiply at low temperatures coupled with the virulence factors it produces (e.g. siderophores, listeriolysin O, phospholipase enzyme and internalins) increases the virulence and/or pathogenicity of the bacterium in human hosts. Complications with L. monocytogenes infection in humans usually occur in pregnant women, and this can lead to stillbirth, premature birth or abortion due to intrauterine infection. L. monocytogenes crosses the placenta and the blood-brain-barrier (BBB), thus causing bacteraemia, sepsis, meningitis and other disseminated infection in neonates or newborns. L. monocytogenes can also be acquired by newborns from the vagina (birth canal) of an infected mother who has a disseminated infection; and manifestation or clinical episodes of the disease usually occur at a later time in the affected infants.

LABORATORY DIAGNOSIS OF LISTERIA MONOCYTOGENES INFECTION

The laboratory diagnosis of L. monocytogenes requires the identification of the pathogen by culture and microscopy. Blood, cerebrospinal fluid (CSF) and focal lesions are the primary specimens obtained from infected patients for analysis. Mueller-Hinton agar and blood agar (BA) are preferably used for the cultivation of L. monocytogenes in the laboratory. L monocytogenes produces small, grayish, haemolytic and translucent colonies on BA. Culture plates are incubated for 48 h at temperature range of 3-40oC. Selective media (e.g. Listeria oxford agar base) also exist for the isolation of L. monocytogenes from clinical and environmental samples.

Growth of L. monocytogenes on chromogenic agar.

L. monocytogenes also grow on Oxoid Brilliance Listeria agar (OBLA) plate causing haemolysis. L. monocytogenes forms beta-haemolysis on blood agar, and its rods resemble corynebacteria. The isolation of L. monocytogenes is enhanced if the specimen is kept for some number of days in the refrigerator (at 4oC) before culturing. L. monocytogenes is Gram-positive, catalase positive, and motile; and its colonies forms beta-haemolysis on blood agar. The pathogen ferments carbohydrates (e.g. glucose and maltose) to produce acid but not gas. Polymerase chain reaction (PCR) and ribotyping are the two molecular techniques employed in the detection of the parasite; and the molecular subtypes of the organism can be detected by pulse-field gel electrophoresis (PFGE).

TREATMENT OF LISTERIA MONOCYTOGENES INFECTION

Sulphamethoxazole-trimethoprim, penicillin G, erythromycin, fluoroquinolones, vancomycin and ampicillin are the drugs of choice for the treatment of listeriosis. Early treatment of infected individuals (including the elderly, pregnant women and the immunocompromised) with any of these agents or a combination of them (according to the guidelines of a physician) is effective in aborting complications due to L. monocytogenes infection. No vaccine currently exists for listeriosis.

PREVENTION AND CONTROL OF LISTERIA MONOCYTOGENES INFECTION

Incidence of L. monocytogenes infections amongst humans could be reduced by taking precautions when consuming some foods. Some type of food meant for human consumption (e.g. salads, sandwiches, vegetables, pork meat, cheeses, milk, and other dairy and fermented food products) can become infected by L. monocytogenes at anytime during food processing and food cultivation. L. monocytogenes is a psychrotolerant bacterial pathogen, and thus refrigeration (i.e. cold temperature) which normally inhibits the growth of most bacterial pathogens is ineffective in limiting its growth.

Proper care should be taken in the handling and processing of food in order to reduce the rate of contamination. As a way of prevention and control of the disease, immunocompromised individuals, the elderly and pregnant mothers should be cautious and avoid unpasteurized dairy and ready-to-eat meats and foods as much as possible since these food sources are the main route via which the pathogen gets transmitted to humans. Food processing companies and food vendors should endeavour to always abide and observe the hygienic principles associated with their businesses so that the rate of transmission of L. monocytogenes could be reduced.

ACTINOMYCETES

Actinomycete comprises a group of Gram-positive, slow-growing, pleomorphic and rod-like anaerobic bacteria that form spores and mycelium (i.e. the branching filaments or hyphae of fungi). They are fungi-like filamentous bacteria. Mycelium formation in actinomycetes usually occurs during the growth of the bacteria, and is scarcely seen in old cultures. Actinomycetes are important normal microflora of the gastrointestinal tract (GIT) of humans and animals; and some are also found in the oral cavity of humans as microflora. They are widespread, and are naturally found in aquatic habitat, composts and in the soil. Morphologically, actinomycetes resemble corynebacteria and mycobacteria. Actinomycetes are distinct from other bacteria species or genera in that they are filamentous organisms, and they also form conidia (spores) and reproduce by asexual reproduction. Most actinomycetes are non-motile organisms. Some of the key genera of actinomycetes include: Nocardia, Actinomyces, Streptomyces, Rhodococcus and Actinomadura.

Nocardia species are aerobic and spore-forming branching bacilli normally found in the soil as saprophytes, and they are implicated as causative agent of nocardiosis in man. Actinomyces species are anaerobic or microaerophilic branching bacilli normally found in the soil, oral cavity, female genitalia and GIT as normal flora, and they are implicated as causative agent of actinomycosis. Streptomyces species are aerobic branching bacilli found naturally in the soil as saprophytes. They rarely cause human disease. Several species of the genus Streptomyces are of industrial and medical importance because they synthesize a wide variety of antibiotics used to treat bacterial infections. For example, tetracycline, streptomycin, clindamycin, neomycin, nystatin and amphotericin B are synthesized by S. aureofaciens, S. griseus, S. lincolnensis, S. fradiae, S. noursei and S. nodosus respectively. Rhodococcus species are aerobic cocci-bacilli organisms commonly found in the soil and in animals (e.g. horses); and they are implicated as causative agents of pneumonia in man. Actinomadura species are aerobic branching bacilli that cause mycetoma in humans.     

IMPORTANCE OF ACTINOMYCETES

Actinomycetes are opportunistic bacteria with much resemblance to fungi organisms because of their ability to form long-branching filaments (mycelium) and conidia or spores. They occur naturally in the soil and in other habitats, and possess some of the following benefits:

  • They are saprophytic organisms.
  • They are the primary source of most naturally synthesized antibiotics used clinically to treat infectious diseases (e.g. streptomycin and tetracyclines).
  • They are human and animal pathogens.
  • Some species also cause disease in plants.
  • They play significant roles in the mineralization of organic matters.
  • Actinomycetes produce secondary metabolites that are of immense importance in the industry, medical and pharmaceutical companies.

 REFERENCES

Prescott L.M., Harley J.P and Klein D.A (2005). Microbiology. 6th ed. McGraw Hill Publishers, USA.

Madigan M.T., Martinko J.M., Dunlap P.V and Clark D.P (2009). Brock Biology of Microorganisms, 12th edition. Pearson Benjamin Cummings Inc, USA.

Goldman E and Green L.H (2008). Practical Handbook of Microbiology, Second Edition. CRC Press, Taylor and Francis Group, USA.

Gillespie S.H and Bamford K.B (2012). Medical Microbiology and Infection at a glance. 4th edition. Wiley-Blackwell Publishers, UK.

Garcia L.S (2010). Clinical Microbiology Procedures Handbook. Third edition. American Society of Microbiology Press, USA.

Abbott, S. L. (2007). Klebsiella, Enterobacter, Citrobacter, Serratia, Plesiomonas, and Other Enterobacteriaceae. In P. R. Murray, E. J. Baron, J. H. Jorgensen, M. L. Landry & M. A. Pfaller (Eds.), Manual of Clinical Microbiology (9th ed., pp. 698-711). Washington, USA: ASM Press.

Balows A, Hausler W, Herrmann K.L, Isenberg H.D and Shadomy H.J (1991). Manual of clinical microbiology. 5th ed. American Society of Microbiology Press, USA.

Barrett   J.T (1998).  Microbiology and Immunology Concepts.  Philadelphia,   PA:  Lippincott-Raven Publishers. USA.

Basic laboratory procedures in clinical bacteriology. World Health Organization (WHO), 1991. Available from WHO publications, 1211 Geneva, 27-Switzerland.

Black, J.G. (2008). Microbiology:  Principles and Explorations (7th ed.). Hoboken, NJ: J. Wiley & Sons.

Brooks G.F., Butel J.S and Morse S.A (2004). Medical Microbiology, 23rd edition. McGraw Hill Publishers. USA.

Murray P.R, Baron E.J, Jorgensen J.H., Pfaller M.A and Yolken R.H (2003). Manual of Clinical Microbiology. 8th edition. Volume 1. American Society of Microbiology (ASM) Press, Washington, D.C, U.S.A.

Murray P.R, Baron E.J, Jorgensen J.H., Pfaller M.A and Yolken R.H (2003). Manual of Clinical Microbiology. 8th edition. Volume 2. American Society of Microbiology (ASM) Press, Washington, D.C, U.S.A.

Murray P.R., Rosenthal K.S., Kobayashi G.S., Pfaller M. A. (2002). Medical Microbiology. 4th edition. Mosby Publishers, Chile.

Talaro, Kathleen P (2005). Foundations in Microbiology. 5th edition. McGraw-Hill Companies Inc., New York, USA.

Taylor LH, Latham SM, Woolhouse ME (2001). Risk factors for disease emergence. Philos Trans R Soc Lond B Biol Sci, 356:983–989.

 

 

Leave a Reply

Your email address will not be published. Required fields are marked *