Introduction, colonisation and polymicrobial interactions
Introduction
Disease in an animal will only occur when there is cell damage or physiological malfunctioning that interferes with the animal’s functions. Pathogenic bacteria, being free-living, have developed a toolbox of strategies that allows them to successfully survive and grow in and on animals. For many bacteria, the animal is an excellent habitat. It is rich in nutrients, warm and provides a moist environment. In sterile sites, it has access to nutrients and no competition with other microbes. However, they must find a way to invade their hosts and then overcome the host’s defences. Exceptions are toxigenic bacteria, where the bacterial toxin is ingested and causes disease without the need for the bacterium to colonise and invade the body. An example is “botox”, a potent nervous system toxin produced by the causative agent of botulism, Clostridium botulinum,
Colonisation on the host, the ability to invade the host and overcome host defences are all considered to be aspects of invasiveness. There are exceptions to this rule, i.e. bacteria such as Borrelia species and Ehrlichia canis are transmitted by ticks. Thus, they will not have to colonise the host, but will directly invade the host through the tick’s biting mouth parts.
The pathogenicity of a bacterium is usually associated with a bacterium’s ability to produce molecules, known as virulence factors, that assist it in subverting or evading the immune response or in causing detrimental physiological changes or essential tissue damage.
The six steps in the pathway most bacteria follow in disease generation or pathogenesis are discussed in Objective 1.
Learning Objectives
- Name the six steps in the pathogenesis of most invasive disease-causing bacteria and fungi.
- Explain the functions of bacterial structures used in the colonisation of the host.
- Contrast the role of bacterial colonisers in health and disease
- Explain the role of bacterial L-forms in disease.
- Explain where and how bacterial biofilms are formed and their role in the pathogenesis of disease.
- Explain the different ways pathogenic bacteria cooperate with other microorganisms to cause disease and relate this to the control of these diseases.
Criteria for disease
Most bacteria are common in animal and environmental ecosystems where they serve to protect animals against disease. However, it is these same bacteria, when allowed to predominate in a population, will cause disease. They are opportunistic. The diagram below shows 6 steps. This is based on Koch's postulates.
Bacterial Epidemiology and Transmission
Epidemiology associated with bacterial disease examines how pathogens are spread and transmitted within and between populations of animals. Epidemiology makes use of statistics and mathematical modelling to predict disease spread and when diseases are most likely to emerge. They often use “Geographical Information Systems (GIS)” which show the spatial distribution of disease. It also statistically determines the risk factors (i.e. changes in climate, animal movement and distribution, farming practices) associated with a particular disease.
For host-adapted microbes, transmission to new hosts must be sustained. The transmission potential of a microbe is mathematically represented as:
R0 = transmission rate due to microbe + host population disease rate + recovery rate.
If R0>1, then microbial transmission can be sustained.
Transmission of pathogens can occur via the oral, respiratory, venereal, contact via damaged or intact mucosae and skin and percutaneously via an arthropod vector, needles or injuries. An understanding of the microorganism, its source, its environmental fragility and point/s of entry allow us to control its transmission.
The quiz at the end of this section has questions for you to test your knowledge on transmission routes.
You will receive more detailed lectures on the epidemiology of disease. Integrate your knowledge of epidemiology with your knowledge of bacterial-host interactions to fully understand the outcomes of a bacterial infection in an animal or population of animals.
Definitions
Bacteriocins – These are proteins produced by bacteria that inhibit or kill closely related bacteria
Contagious – Spread from one individual to another, usually by direct contact
Infection – The invasion of and multiplication in body tissues by disease-causing microorganisms.
Invasiveness – the ability of a micro-organism to invade tissue. To do this, they have to be able to colonise, produce invasive molecules and overcome the innate and adaptive immune response of the host.
L-form – Living bacteria that have temporarily stopped producing an outer membrane.
Microbial pathogenicity – the structural and biochemical mechanisms whereby microorganisms cause disease.
Minimum Infective Dose – This is the number of microbes required at a surface for them to invade or infect the host and cause disease.
Virulence – Degree of damage caused by a microorganism
Virulence factors – Structures or molecules produced by micro-organisms that assist them to subvert or evade the immune response and cause detrimental physiological changes or essential tissue damage
Test for knowledge of definitions using the following quiz.
Colonisation
Step 1: Reach the target cells
Prior to the process of colonisation, bacteria must be able to reach their target cells that are usually found on body surfaces. Bacteria make use of a number of strategies, including:
1. Animal behaviour, i.e. nose greeting and licking or fighting and natural processes of the animal, i.e. swallowing and intestinal peristalsis
2. Bacteria use their own structures to find their target cells. In fluids, motile bacteria make use of flagella that propel them to the target cells. i.e. the zoospores of Dermatophilus congolensis have a single flagellum that propels them forward using a whip-like motion and chemical receptors that detect carbon dioxide emitted from skin. Watch this YouTube video [2:13] about non-motile and motile bacteria using flagella. Some bacteria have flagellae integrated in their outer membrane that allow them to snake their way through the viscous mucus to reach target cells, i.e. the spirochaete Leptospira.
3. Some bacteria, like the wall-less bacteria, have steroid-rich membranes that are super-flexible, giving them amoeba-like movement abilities.
4. Epithelia like the intestinal tract are covered in mucins, immunoglobulins and antibacterial peptides. These trap the bacteria in the upper mucus level and prevent them from coming into contact with the membranes of epithelial cells. Intestinal peristalsis will constantly remove trapped bacteria. Some bacteria will produce enzymes that dissolve mucus and other substances to reach their target cells.
Some bacteria can avoid the colonisation step if they are provided with a portal of entry, i.e. wounds or invertebrate (ticks, lice, flies) bites.
Step 2 Colonisation
Colonisation is the ability of bacteria to attach to cell surfaces on the host at the portal of entry.
It usually involves two processes: docking, where the attachment is reversible and non-specific and then the more intimate and permanent attachment of anchoring, where ligands of bacterial adhesins will bind to their complementary receptors on host cells (specific attachment). Bacterial adhesins include flagellae, fimbriae (common pili), the glycocalyx that consists of polysaccharide capsules and slime, and outer membrane (wall) proteins. Cell receptors are usually glycoproteins.
- Docking often involves electrostatic or hydrophobic forces. Bacterial biofilms produced by bacterial communities are hydrophobic, allowing bacteria to adhere to body surfaces and protecting them from the animal’s protective mechanisms.
- Anchoring to cell surfaces allows bacteria to resist wash-out (the actions of host cilia, mucus and peristalsis). It also means that certain bacterial species and even different strains of a bacterial species will be host and site specific = ligand-receptor interaction. To do this, bacteria use pili and outer surface membrane proteins, known as adhesins. For example, the K88 pili on pathogenic strains of Escherichia coli will only bind to intestinal cell receptors of pigs that have receptors for these fimbriae, and E. coli with P-fimbriae are able to attach to the epithelium of the urinary bladder. Instead of being one of the more common diseases of weaned piglets, colibacillosis or post-weaning scours in piglets could be a disease of the past if we could breed piglets free of this receptor. (Piglets do lose this receptor with age, so colibacillosis is a disease of piglets, not older pigs.) Some bacteria may inject molecules into cells that stimulate the cells to alter their surface structure, improving bacterial adhesion to cell surfaces. “Attaching and effacing” strains of Escherichia coli inject “Tir”, a protein, into the epithelial cell that stimulates the actin in the plasma membrane to remodel to form a pedestal. Tir forms a strong bond with intimin on the surface of the E. coli.
- Attaching and effacing E. coli docks onto the mucin layer, and injects Tir protein into the cell. Tir moves to the plasma membrane and stimulates host actin to remodel the membrane to form pedestal structures. The Tir forms a strong bond with bacterial surface intimin, affording a very strong attachment for E. coli. This action results in decreased nutrient and fluid absorption by affected cells.
Bacterial interactions in health and disease
Under natural circumstances, bacterial species colonising body surfaces will co-exist and interact in a complex environment. In the animal, these communities can be found on the skin, gastrointestinal and upper respiratory tracts, and external genitalia. Internal body structures are essentially sterile.
In healthy individuals, resident microbial populations are stable and contribute to the good health of their host. Endogenous microflora provides host nutrients, including vitamins, they “train” local immunity, competitively exclude pathogens and assist in digestion.
In people, the normal microflora have been shown to be protective against irritable bowel syndrome, diabetes type 2 and obesity. They also affect our “moods”. People suffering from Crohn’s disease, a severe hyperimmune response, within the intestinal tract, have benefited from the administration of fresh faeces from healthy members of their household. This is known as a probiotic.
What if the endogenous microflora are disrupted?
Disruptions to endogenous microflora, such as antibiotic use, changes in nutrition, can damage to epithelial barriers and immune-compromise, encourage overgrowth of opportunistic pathogens. This is often called dysbiosis.
In a healthy rumen, the predominant microflora are obligate anaerobic bacteria, fungi and protozoa that produce fatty acids when digesting cellulose and lignins. Ruminants cannot digest plant material and use the bacterial manufactured fatty acids as an energy source. When the diet of the ruminant is suddenly changed to a predominantly carbohydrate ration, i.e. a diet rich in grains, the smaller portion of the rumen microflora that are sugar fermenters start to predominate. Their more acidic products, such as lactic acid, decrease the rumen pH. This will kill the normal microflora as well as damage/ “burn” the rumen epithelium. Pathogenic bacteria and fungi that are able to withstand the acid environment are then able to proliferate in the rumen and invade the body through the damaged mucosae.
Broad-spectrum antibiotics given orally or those that undergo hepatobiliary excretion can destroy susceptible commensal bacteria in the intestinal tract and result in “dysbiosis”. Horses are especially susceptible to the adverse effects of antibiotics and develop diarrhoea and colic (abdominal pain).
Bacterial L-form bacteria or cell wall deficient bacteria
A special case: Bacterial L-form bacteria are either gram-positive or gram-negative bacteria that, after exposure to antibiotics like penicillin that target cell outer membranes, antibacterial enzymes (lysozyme), and bacteriophages, have stopped producing outer cell membranes. In fact, they are able to destroy their own outer membranes through the production of endolysins. While not having an outer membrane makes them susceptible to osmotic shock, they are able to survive in isotonic watery environments such as the intestinal, urinary and reproductive tracts. They are more common in chronic infections. They again produce an environmentally resistant outer cell membrane when the inhibitors have been depleted. L-forms tend to be spherical in shape and divide by pinching off a portion of their cell. I have encountered them in persistent urinary tract infections.
Biofilms and disease
Biofilms are communities of bacteria, either as a single species or several species that adhere to surfaces and produce a profuse, protective extracellular polymer substrate/matrix (EPS).
A biofilm forms in a multistep process:
- Bacteria loosely adhere to suitable surfaces via their hydrophobic surfaces in the process known as docking. It is reversible – can be washed off. Adherence is facilitated by the presence of flagella. Surfaces that are rough and in contact with fluids are more prone to biofilm colonisation.
- Once bacteria have anchored (more stable adherence) to a surface and have enough nutrients to grow, they will increase, forming microcolonies.
- They produce extracellular polymer substrate/matrix (EPS). This matrix hydrates and nourishes embedded bacteria and protects them from destruction from factors within their environment, i.e. host immune factors.
- The smaller biofilms can now develop into larger complex colonies. Bacteria deep within the EPS receive less nutrients and thus tend to become metabolically inactive which prolongs their lifespan and makes them resistant to attack by antibiotics and cytokines. In this vegetative state, they remain localised and tend not to be invasive or virulent. However, they do act as a source of pathogens and can persist within the host. Since the bacteria are in close contact, the transfer of genetic material, including mutations conferring antibiotic resistance or virulence factors, is common. They then communicate with each other via quorum sensing. Bacteria in biofilms produce autoinducers which increase as the bacterial population increases. This allows a coordinated action between communities i.e. they will up-regulate or down-regulate certain genes, i.e. for antibiotic resistance or some virulence factors, increase EPS production or even bioluminescence.
5. During maturation of the biofilm, sessile bacteria at the surface will disperse. Bacteria in a mature biofilm will occasionally break off due to shearing forces and become planktonic. This is only significant clinically when large numbers of bacteria become planktonic. This happens when conditions change. For example, when iron becomes available, there is a drop in c-di-GMP (nutritional stress response by bacteria), bacteria in a urinary biofilm will disperse. They will then become fully active, setting up new infections.
Examples of where biofilms occur on animals:
- mouth and teeth
- urinary bladder
- bladder stones
- heart valves
- indwelling catheters.
All the biofilms discussed were on cell surfaces; however, there is an increasing body of evidence that bacteria also produce intracellular biofilms. In persistent canine urinary tract infection, uropathogenic Escherichia coli (UPEC) enters bladder cells, escapes the phagocytic vesicle and divides, forming packed clusters of cocci that are encased in a mass of fibres. As they grow, they push against the side of the cell, causing it to protrude. The bacteria elongate but don’t divide, forming long filaments. These then enter the bladder lumen and infect another bladder cell.
Importance and therapeutic implications of biofilms
- Persister bacteria (nutritionally deprived, metabolically inactive bacteria at the base of a biofilm (blue bacteria below) are long-lived and usually non-responsive to antibiotics and are a reservoir of pathogenic bacteria.
- Bacteria in a biofilm are close and can access DNA from the EPS via transduction and from other bacteria through conjugation.
- The EPS acts as a barrier to antibody and antibiotic access to bacteria. The efficacy of antibiotics in a biofilm can be decreased up to 1000 times.
Possible therapeutics to reduce biofilms
Combining some of the methods below will yield the best results.
- If possible, mechanical removal of biofilms, i.e. wound debridement, ear cleaning, removal of indwelling catheters. Fine water sprays and ultrasound can disperse superficial biofilms.
- Combinations of biofilm destroying chemicals and antibiotics, i.e. acetylcysteine and linezolid (an antistaphylococcal antibiotic)
- Bacteriophages – bacteria-specific viruses that destroy bacteria even if they are in the stationary phase.
- Silver nanoparticles – these cations attract bacteria and are taken up by bacteria. They inactivate bacterial enzymes. Their large surface area to small size allows them to be dosed with small doses below the toxic threshold. They are commonly used in wound dressings. Gallium has also been used.
- Honey on wounds. Maluka honey is the most effective honey. It prevents bacteria from adhering to wounds.
- Mineral chelators, i.e. EDTA. Lactoferrin, an iron chelator found in milk, can decompose bacterial biofilms.
- Biofilm dispersal proteases and nucleases, i.e. glycoside hydrolases, lysozyme
- Broad-spectrum cationic peptides that target c-di-GMP (nutritional stress response by bacteria)
- Struvite stones in the urinary bladder are produced by the action of urease produced by biofilm bacteria on urea to induce an alkaline environment needed to decrease the solubility of minerals which cause mineralisation around the biofilm. Treatment with a urease inhibitor and antibiotics has a better clinical cure than antibiotics alone.
- Cold atmospheric plasma. Ionised gas, including helium, argon, nitrogen or air, is used to disrupt biofilms and kill bacteria. It works by generating reactive oxygen and nitrogen species which are antiinflammatory and antimicrobial and can penetrate biofilms.
Micro-organism interactions in Disease
Although micro-organisms can work against each other in a complex community (see previous section), there are those that work collaboratively to encourage the growth of pathogens by providing a suitable environment; uncoating host receptors, damaging epithelia and diverting or suppressing the immune system.
Viral-bacterial INTERACTIONS IN DISEASE
You will learn that bacteriophages can destroy bacteria. However, some known as lysogenic phages can enhance the pathogenicity of bacteria in several ways, including horizontal gene transfer, biofilm production, immune evasion and virulence. You will encounter several examples of these.
Many infectious diseases are multi-factorial in aetiology, where viruses or other infectious agents enhance the colonisation and growth of opportunistic bacterial pathogens. The best known examples are diseases of the upper and lower respiratory tracts.
It is now known that fatalities in the devastating influenza pandemic post 1st World war in 1918 was due to secondary bacterial pathogens such as streptococci and staphylococci.
How do viruses assist bacteria in colonisation and invasion?
1. Impede mucociliary clearance. Increased mucus is produced to rid the airways of viruses, however, excessive mucus, especially if it is dehydrated, will block the airways, reducing mucociliary clearance. Some viruses alter the activity and number of cilia, altering the upwards beat of the cilia. Some bacteria like mycoplasmas, the wall-less bacteria, and Bordetella bronchiseptica paralyse or destroy the cilia causing the same effect.
2. Alter surface receptors. Some viruses cause certain host proteins to be more expressed on cells. For example, respiratory viruses can increase the expression of surface fibronectin to which pathogenic bacteria like streptococci adhere to. Some viruses will express their own proteins that will act as a linking molecule for the adhesion of bacteria to cells.
3. Damage epithelia. Viral replication and inflammation in response to viral infection provide a portal of entry and nutrients for bacteria
4. Alter immune responses. The immune system must always guard against an excessive immune response. To prevent over-activation of the immune response to viruses, a negative feedback mechanism can lead to local immunity being refractory to responding to secondary bacterial infections. Respiratory viruses can also utilise all the local immune cells or inhibit their activity, i.e. alveolar macrophages.
Conversely, bacteria can reveal surface adhesion sites for viruses as well as divert the immune system, allowing viruses to more easily access the respiratory tract. They can also destroy viral receptor sites and decrease the viral load. Many viruses use components of the bacterial outer membrane to stabilise themselves from heat stress.
Bacterial-bacterial INTERACTIONS
Bacteria in polymicrobial communities not only compete with each other but will also act collaboratively. Already discussed is how bacteria interact in biofilms.
Example 1: Bacterial interaction in ovine footrot
Another good example of bacterial-bacterial interactions occurs between obligate anaerobes and facultative anaerobes. The facultative anaerobes consume all the oxygen radicals in the local environment, protecting the obligate anaerobes from oxidative damage. This typically occurs in the oral cavity and the rest of the gastrointestinal tract. An interesting case of mutualism occurs in the ovine foot when the facultative anaerobe Trueperella pyogenes mops up the oxygen radicals, allowing the proliferation of Dichelobacter nodosus and Fusobacterium necrophorum. Fusobacterium necrophorum produces a locally active leucotoxin which destroys neutrophils, allowing the survival of the other 2 bacterial species. Virulent strains of Dichelobacter nodosus produce a protease that is able to destroy hoof lamellae and provide a source of nutrition for the other two bacteria.
Example 2: Bacterial interaction in porcine adamatosis
Lawsonia intracellular, the agent of porcine intestinal adamatosis, will only invade the epithelium and cause proliferation of the ileum of growing pigs when there is normal microflora present
Example 3: Bacterial interactions in salmonellosis and colibacillosis
In the intestine, another interesting interaction occurs. Some of the intestinal microbiota produce hydrogen sulphide that is converted to thiosulphate by the enterocytes. During inflammation, neutrophils recruited to the intestines release reactive oxygen species (ROS) that oxidise the thiosulphate to tetrathionate. Salmonella enterica, an enteropathogen, is able to anaerobically respire tetrathionate. Anaerobic intestinal bacteria also release enzymes that are able to liberate nutrients such as fucose and sialic acid from intestinal cells that nourish other microbiota, including the enteropathogens. Pathogenic Escherichia coli (EHEC) upregulate their expression of virulence genes in the presence of large quantities of fucose.
Bacterial-nematode INTERACTIONS
Annual rye grass toxicity
During the period of seeding, the ingestion of corynetoxins found in the seeds of annual rye grass (Lolium rigidum) causes disease of the nervous system in cattle, sheep and horses. These toxins are produced by Rathayibacter toxicus, a soil bacterium that forms biofilms on the cuticle of a nematode known as Anguina funesta. The larvae of this worm infest the ryegrass seeds, causing gumming disease. The bacteria grow in the gum, producing corynetoxins. These toxins are ingested and have a cumulative effect, inhibiting protein glycosolation, leading to damage of the liver and central nervous system of the animal. Here is a link to a video of horses suffering from ryegrass staggers.
Note that animals ingesting perennial ryegrass can develop similar clinical signs due to Penitrem A, a fungal toxin. A link to a video of alpacas with this toxicity.
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Opportunistic pathogens are those that are usually present in the animal or in its environment that only cause disease when the host's resistance to disease has been altered.
These are 4 postulates published by Robert Kock in 1890. Due to the multifactorial nature of some diseases, they are no longer validate for all diseases. However, they do provide a good framework for understanding infectious disease.
The establishment of micro-organisms at the portal of entry into the host. This is usually on surfaces such as all mucosae and the skin.
Adhesins are cell-surface components or appendages of bacteria that facilitate adhesion or adherence to other cells or to surfaces, usually in the host they are infecting or living in
Temporary surface attachment by bacteria using electrostatic or hydrostatic forces
Biofilms are communities of bacteria, either as a single species or several species that adhere to surfaces and produce a profuse, protective extracellular polymer substrate/matrix (EPS).
Firm attachment of bacterial structures to host tissue surfaces
Having an internal cause or origin.
Are medicines that either kill or inhibit bacteria
These are micro-organisms that only cause disease when given an opportunity i.e. when the host has a weakened immune system or when mucosal or skin barriers are damaged. They are often part of the commensal microflora of the host.
Destruction or disruption (imbalance) of the normal microflora on or in the body. Example: It occurs when there is broad-spectrum antibiotic therapy
Cell-to-cell communication by bacteria through the production of signaling molecules known as autoinducers.
This is when "foreign" DNA is introduced into a bacterium by a bacterial virus known as a bacteriophage.
This is when organisms exchange genetic material via direct contact with each other. Bacteria use the pilus as a bridge.