Introduction to microbiology

Question 1

Describe Koch’s postulates and list some problems and limitations of Koch’s postulates

Answer 1

Koch’s postulates

• A micro-organism must be found in abundance in all organisms suffering from the disease, but should not be found in healthy organisms*
• A micro-organism must be isolated from a diseased organism and grown * in pure culture
• A cultured micro-organism should cause disease when introduced into a healthy organism*
• A micro-organism must be re-isolated from the inoculated, diseased experimental host and identified as being identical to original specific causative agent

• Depending on the strength of the immune system of cell or host, a micro-organism may not need to be in abundance in order to cause disease
• Additionally disease-causing organisms may be present in otherwise healthy individuals (i.e. in asymptomatic/subclinical cases e.g. polio, herpes, HIV, hepatitis C, cholera, typhoid, SARS-CoV-2, or commensal bacteria that only cause disease in immunocompromised individuals or when in certain locations)
• Some micro-organisms that are capable of causing disease cannot be cultured e.g. prions and Creutzfeldt-Jakob disease
  (Side not: technically speaking prions are not micro-organisms, see ==prions==)
  -
• Not all organisms exposed to the infectious agent will acquire the infection

This just serves to demonstrate the complexity of microbial disease, as well as to show how our understanding of microbiology has evolved over time.

Question 2

Distinguish between microbiology and medical microbiology

Answer 2

Microbiology is the study of small, living things, whereas medical microbiology is the prevention, diagnosis and treatment of infectious diseases in humans.

Question 3

List the types of microorganisms associated with human diseases

Answer 3

The scope of microorganisms associated with human disease is broad and varies in both size and complexity. Parasites, fungi, bacteria, viruses and prions ^[algal infections known as protothecosis are rare] are the focus of medical microbiology.

Question 4

Define parasite and describe their features

Answer 4

Parasites are the most complex microbes. They are eukaryotic organisms, and can be classed as protozoa or metazoa, depending on whether they are unicellular, or multicellular, respectively.

The lifecycles of parasites can range from relatively simple to complex. Examples include beef and pork tapeworm, in which the adult stage has a singular, definite host (humans). A more complex example is cryptosporidium, where development occurs in humans as well as the environment.
An even more complex life-cycle is that of Plasmodium, which includes many stages of development, asexual and sexual cycles.

Parasites can also vary in the sites they occupy within the body i.e. whether infections are exclusively extracellular, as is the case with helminths and arthropods, or whether infection occurs intracellularly and/or extracellularly such as with infections of protozoa.

Understanding the parasitic lifecycle is important to understand disease transmission, incubation period, clinical symptoms, diagnostic specimens and tests, and means of prevention and treatment

Question 5

Describe fungi and their features

Answer 5

Fungi are eukaryotic organisms. They possess a rigid cell wall composed of chitin and glucan. ^[As these features differ from human, they constitute a therapeutic target]
Like animal cells, fungi also possess cell membranes, except with ergosterol^[ibid] as the major sterol instead of cholesterol.

Fungi can exist as unicellular yeasts. These reproduce by budding or fusion; in other words, asexually. They resemble bacteria in their morphology.

Fungi can also exist as multicellular moulds. Their morphology is filamentous, with characteristic features such as hyphae and conidia. They are said to appear fluffy. Moulds can produce spores which are infectious. Additionally, moulds are capable of reproducing asexually as well as sexually. As moulds are capable of releasing spores, they are considered the ‘infectious form.’ This is another distinction between yeast and mould.

Unicellular yeast | Multicellular mold
:———————-:|:————————-:
![[yeast.jpg]] | ![[mold.jpg]]

Additionally, some fungi are capable of switching between yeast form and mould form — in other words they are said to be dimorphic. This ‘switching’ may be environmentally influenced e.g. at body temperatures, a fungus will exist as a yeast.

Fungal infections can be classified based on the site of infection i.e. if the infection is superficial, cutaneous or subcutaneous, or a systemic/deep mycoses. Systemic mycoses often occur in immunocompromised or hospitalised patients— thus immunosuppression is a major risk. High mortality rate is associated with systemic mycoses.

Note that fungi are causative agents of other, non-infectious diseases. They can produce toxins such as the Amanita mushroom, and constitute allergens indicated in allergic rhinitis and allergic bronchopulmonary aspergillosis.

 Superficial mycoses are limited to the stratum corneum and essentially elicit no inflammation. Cutaneous infections involve the integument and its appendages, including hair and nails. Infection may involve the stratum corneum or deeper layers of the epidermis. Inflammation of the skin is elicited by the organism or its products. Subcutaneous mycoses include a range of different infections characterized by infection of the subcutaneous tissues usually at the point of traumatic inoculation. An inflammatory response develops in the subcutaneous tissue frequently with extension into the epidermis. Deep mycoses involve the lungs, abdominal viscera, bones and or central nervous system. The most common portals of entry are the respiratory tract, gastrointestinal tract, and blood vessels
 (https://www.ncbi.nlm.nih.gov/books/NBK7902/)

Question 6

Give an example of a parasite

Answer 6

helminths - extracellular parasites

intracellular/extracellular - Plasmodium falciparum

Question 7

Give an example of a fungus

Answer 7

Candida
- found in the mouth
-endogenous/commensal pathogen that has the potiential to become pathogenic when antibiotics kill normal (bacterial)f lora of vagina: resulting in vaginal thrush

Question 8

Describe the different ways in which fungi are classifed

Answer 8

Fungi can be classified on the basis of morphology i.e. if they are unicellular mould or multicellular moulds.
Similarly, fungal infections can also be classified, based on the site of infection i.e. if the infection is superficial, cutaneous or subcutaneous, or a systemic/deep mycoses. Systemic mycoses often occur in immunocompromised or hospitalised patients— thus immunosuppression is a major risk. High mortality rate is associated with systemic mycoses.

Question 9

Explain the different morphologies seen in yeast

Answer 9

Fungi can exist as unicellular yeasts. These reproduce by budding or fusion; in other words, asexually. They resemble bacteria in their morphology.

Fungi can also exist as multicellular moulds. Their morphology is filamentous, with characteristic features such as hyphae and conidia. They are said to appear fluffy. Moulds can produce spores which are infectious. Additionally, moulds are capable of reproducing asexually as well as sexually. As moulds are capable of releasing spores, they are considered the ‘infectious form.’ This is another distinction between yeast and mould.

Additionally, some fungi are capable of switching between yeast form and mould form — in other words they are said to be dimorphic. This ‘switching’ may be environmentally influenced e.g. at body temperatures, a fungus will exist as a yeast.

Additionally, some fungi are capable of switching between yeast form and mould form — in other words they are said to be dimorphic. This ‘switching’ may be environmentally influenced e.g. at body temperatures, a fungus will exist as a yeast.

Question 10

Explain why moulds are considered the infectious form of fungus

Answer 10

Moulds can produce spores which are infectious. Additionally, moulds are capable of reproducing asexually as well as sexually. As moulds are capable of releasing spores, they are considered the ‘infectious form.’ This is a distinction between yeast and mould.

Question 11

Compare and contrast yeasts and molds

Answer 11

• yeasts reproduce by budding i.e. asexually
• they are unicellular
• they resemble bacteria in their morphology
• moulds are multicellular
• their morphology is filamentous, with features such as hyphae, and conidia
• appear “fluffy”
• moulds can produce infectious spores
• moulds are capable of reproducing asexually as well as sexually

Question 12

Describe the different ways in which fungi cause disease

Answer 12

• Infectious disease:
  
  • superficial, cutaneous or subcutaneous, or a systemic/deep mycoses. Systemic mycoses often occur in immunocompromised or hospitalised patients— thus immunosuppression is a major risk. High mortality rate is associated with systemic mycoses.
• Non-infectious disease:
  
  • They can produce toxins such as the Amanita mushroom, and constitute allergens indicated in allergic rhinitis and allergic bronchopulmonary aspergillosis.

Question 13

Describe bacteria and their basic features

Answer 13

Bacteria are unicellular, prokaryotic organisms. As such they are relatively simple in structure. They have a cell wall, but do not possess any membrane bound organelles, including the lack of a membrane bound nucleus (instead, a nucleolus is present). Bacteria usually measure between 1-20 um, and reproduce by asexual division.

The structure of bacteria and the composition of their cell walls can differ ^[[Microbiology lecture 3]]
Typical bacterial cell walls contain peptidoglycan. The thickness of peptidoglycan in cell walls can be used to classify bacteria. ^[ibid]

• Gram positive bacteria have cell walls with thick peptidoglycan.
• Gram negative bacteria have thin peptidoglycan in their cell walls

Note:
Cell walls of bacteria can be rich in mycolic (fatty) acids, or alternatively, may be atypical and possess no peptidoglycan.
Bacteria with cell walls rich in fatty acids include Mycobacterium tuberculosis.
Atypical cell walls can be found in Mycobacterium pneumoniae and C. trachomatis.

Bacteria come in a variety of shapes. These include round/coccus–which can be found in clusters or linear chains; spiral; or rod-shaped bacillus.
Knowing structure of bacteria assists in identification of the disease-causing agent i.e. the pathogen, and guides management. ^[Cell wall is responsible for bacteria’s characteristic shape, thus is essential to function]

Question 14

Give examples of Gram positive and Gram negative bacteria

Answer 14

• Gram positive bacteria: - Gram positive bacteria have cell walls with thick peptidoglycan. Examples of gram positive bacteria include Staphylococcus aureus, Streptococcus pneumoniae, and L. monocytogenes. They stain blue or purple
• Gram negative bacteria: E.coli, Neisseria meningitidis, and syphilis. They stain pink

Question 15

Describe the ways in which bacteria can be classified

Answer 15

Shape: i.e. bacillus, coccus, others e.g. filamentous (can occur anomalously e.g. E.coli, or induced in reponse to certain PBP antibiotics), helical (H. pylori), spirochete (Syphilis), corkscrew ( )

Gram staining status

Phylogenetics/evolutionary relationships

Question 16

Describe viruses and their features

Answer 16

Viruses are the smallest infectious particles, with a size range of 18 to 300 nanometres. They may have DNA or RNA as their nucleic acid, sometimes with additional proteins required for replication and pathogenesis.

Viruses are enclosed in a protein coat termed a capsid. Some viruses also possess an outer lipid envelope. Non enveloped viruses are said to be naked. Viruses are obligate parasites, i.e. they require host cells for replication. The cell type is specific for the virus and will determine the symptoms experienced by the host.

Question 17

List the broad steps that viruses take to infect a host

Answer 17

• • attachment
• penetration
• uncoating
• replication
• assembly
• release

Question 18

List and describe the different outcomes of viral infection

Answer 18

The outcomes of a viral infection are varied, and dependent on the virus as well as the host cell’s response. An infection can lead to transformation and tumorigenesis (HPV), a lytic infection (flu), a persistent infection (hepatitis B), or a later infection i.e. on e which later emerges as a lytic infection (herpes simplex hiding out in neural ganglia).

Note that relatively low grade infections can result in more serious complications such as hepatitis B and the subsequent cirrhosis, resulting in increased risk of HCC.

Question 19

Give examples of viruses associated with the different outcomes of infection

Answer 19

• HPV and tumorigenesis
• Flu and lytc infection
• Hepatitis B and persistent infection
• Latent infection and Herpes simplex

Question 20

Describe prions

Answer 20

Prions are proteinaceous infectious particles.
Prions were once known as ‘unconventional slow viruses’; it was unclear what the causative agent was. The prion protein was isolated in the 1990s.

Prion diseases are neurodegenerative — a misfolded protein in the brain induces misfolding of neighbouring proteins. Misfolded proteins form amyloid aggregates which destroy cells and leave behind characteristic holes.

Prion aggregates are stable and are thus resistant to denaturation to standard physical or chemical agents, i.e. are typically resistant to extremes of pH and temperature . In other words, ordinary disinfection methods do not work.
This complicates disposal and containment measures; indeed, it has previously been observed that cases of prion disease have been transmitted iatrogenically by way of infected medical instruments.

> ##### Iatrogenic Prion Diseases

Iatrogenic CJD was first described in 1954 in a patient who received a corneal transplant. A number of cases have since been recorded of iatrogenic transmission of prion diseases, through the use of neurological instruments, transplantation of [dura mater]…

There is no treatment for prion disease and is thus always fatal. Luckily, prion disease is rare.

Question 21

Describe iatrogenic prion disease and how theu spread

Question 22

Explain the importance of clinical microbiology in medical practice

Answer 22

As a doctor, you will need to study microbiology for several reasons:
- you will need to diagnose and treat patients with infections: infections are common and inescapable
- you will have patients with conditions that make them prone to infections e.g. diabetes, HIV, CF, pregnancy
- you will do things (procedures, administering medicines) that put patients which put them at risk of infection e.g. surgery, antibiotics, immunosuppressive drugs, urinary catheters
- you will want to protect patients and the greater public from infection e.g. vaccination, outbreak control by public health physicians

Question 23

Explain what doctors need to know about microbiology

Answer 23

1. Normal flora
   There are about 10^(13) cells in the body, with 10^14bacterial cells associated with them. The normal flora is the population of micro-organisms routinely found growing on the body of a healthy individual. These include resident flora — which inhabit body sites for extended periods of time and re-establishes if disturbed; and transient flora — which inhabit body sites for short periods of time e.g. hours, days, weeks.

The ‘good’ micro-organisms may be alternatively known as microbiota, microflora or simply flora, commensals, endogenous bacteria or normal bacteria.

The relationship between humans and the normal flora is considered to be commensal.
> This refers to a type of symbiosis where one micro-organism gains from the association while the organism (in this case, a human) is neither harmed nor benefited. However, as the human microbiota are considered important for health, the relationship is more aptly described as a ‘mutualistic’ relationship

The normal microflora are found in many sites in the body. These include skin e.g. S. aureus, P. aeruginosa of scalp , nose e.g. S. aureus, mouth e.g. Candida, lung, intestine, urethra, vagina e.g. G- rods, throat e.g. S. pneumoniae, and teeth.

It is important for doctors to understand normal flora in order to interpret microbiology results, i.e. if something grows from a specimen — it is important to determine whether it is normal flora or pathogenic.
It is also necessary in order to understand when normal flora can turn from ‘good’ to ‘bad’ i.e. endogenous pathogens.
Examples include vaginal thrush (Candida) while on antibiotics, intra-abdominal infection due to ruptured appendix, and bloodstream infection (e.g. normal skin flora) from intravascular catheter.

The questions that need to be answered ahead of time, i.e. based on your acquired knowledge, when assessing common pathogens include:
- where do they normally lie i.e. are they endogenous or strictly pathogenic (exogenous)?
- how do they get in to cause infection?
- how do they replicate and disseminate?
- what sort of diseases do they cause?
- how do they spread to others?

There are several ways in which bacteria replicate and disseminate. These include:
- intracellular, where the micro-organism replicates within host cells e.g. all viruses, some protozoa and several bacteria
- extracellular, where micro-organism does not invade host cells, but replicates in extracellular spaces e.g. all helminths, some protozoa and several bacteria ^[note that these bacteria are often more easily cultured]

Additionally, some micro-organisms can disseminate from a primary infection site to secondary site(s) within the body

Several features:
- pathogenic: capable of causing disease
- virulence: degree of pathogenicity of the organism i.e. how likely is the organism to be pathogenic/cause disease?
- virulence factors: the components of a pathogen that, when deleted, specifically impairs virulence but not viability. ^[note that virulence is not solely related to virulence factors alone, but is also related to human/host factors]
Examples of virulence factors:
- adhesin: coordinates binding of organism to host
- invasin: enable organism to invade host cell (characteristic of intracellular pathogens)
- impedin: enables organism to avoid one or more of the host’s defence mechanisms
- aggressin: causes damage to host
- modulin: induces damage in host indirectly

*Virulence factors are important in causing disease
- not all pathogens have all types of virulence factors
- a pathogen may have multiple factors within a category, some individual virulence factors may be classified in multiple categories
n.b. the interaction with the host immune system is also important in determining disease outcomes

3. Host-pathogen interactions

Host’s physical barriers and immune system protect against infection <—> Host’s immune response to infection may contribute to pathogenicity of infection
- Host response starts basic and general, with physical and chemical barriers including normal flora ^[this can be perturbed by health measures. This makes infection prevention key in health services], escalating to more specific innate and adaptive immune systems

Flu

4. Common micro-organisms associated with clinical syndromes
   Several questions must be asked of common micro-organisms associated with clinical syndromes:
   - what pathogens are associated with a particular clinical syndrome e.g. UTIs, pneumonia
   - how do you make a diagnosis?
   - what antibiotics may you need to use empirically while waiting for further results?
5. Diagnostic tests
   The choice of diagnostic test is determined by several factors, including:
   - what type of specimen
   - when to collect the specimen
   - what test to order
   - how to interpret the results

Another consideration is the limitations of the test:
its sensitivity and specificity, negative and positive predictive values.

6. Vaccination and antimicrobial therapy
   This encompasses how to avoid infections e.g. vaccination, malarial prophylaxis, and food preparation, and microbials including main types of antibiotics/antivirals/antifungals/antiparasitic drugs, the spectrum of activity, dosing and common side effects.
7. Basic infection control and public health
   Knowledge of the epidemiology of infectious diseases informs infection control and public health. Considerations include:
   - mode of transmission – contact, airborne, body fluids, food and water
   - incubation period — time from infection to symptoms
   - duration of infectivity
   - methods to prevent infection