Basic Microbiology

Basic Microbiology. The central principle of infection control is that of control of the growth of microorganisms which potentially cause us harm. To underpin the concepts of infection control it is useful to understand some basic microbiology regarding the types of organisms involved, how they may grow and multiply, and the kinds of diseases they may be responsible for. Basically there are three groups of micro-organisms: • Bacteria • Fungi • Viruses

Bacteria. There are many different types of bacteria in existence, and by far the greater percentage of them are harmless to us. Bacteria are also essential to our daily lives, ensuring that we remain healthy, helping the development of drugs, and also in the development of new foods. Bacteria can be grouped visually, depending upon their shape and arrangement : • Cocci – all round or spherical • Bacilli – rod-shaped • Spiral-shaped. (coccobacillus falls between coccus and bacillus). Each group may be further sub-divided : Cocci Monococcus –arranged singly Diplococcus –2 together Staphylococcus –haphazard Streptococcus –chains Sarcina – clumps in cubes Gaffkya –groups of four Staphylococcus is found normally in the body in three formss. aureus in the nasal passages s. epidermis on the skin s. albus in the mouth. Neisseria are similar to diplococcus, but are kidney-shaped. These are usually found in pus, i.e. N. gonorrhoea, N. meningitides.

Bacilli Found as single rods or as chains. Single rods are randomly arranged, and may be either long and thin or short and fat. E. coli is an example, normally found in the bowel. Salmonella is another example.

Spiral. Spirilla –move via flagella Vibrio – Spirochetes –

Structure of bacteria.

The cell membrane of the bacteria is differentially permeable, and is also the site where enzymes occur, sited on the mesosomes. The cell wall determines the shape of the bacteria, and bacteria can also be classified by how they take up the Gramm stain, which is dependent upon the structure of the cell wall. This occurs because all bacterial cell walls contain muerin, a peptidoglycan. Muerin consists of parallel strands of polysaccharide, cross-linked by short polypeptide chains to give a rigid structure, forming a net around the bacteria. In Gramm positive bacteria the cell wall has a muerin region thicker than Gramm negative bacteria. The individual layers of muerin are connected by peptide cross bridges. This forms a very rigid framework for the cell wall. Teicoic acid (another polysaccharide) is also associated with the cell wall, and we utilise this to make it possible to identify the bacteria by immunological means. Gram negative bacteria contain less muerin and have no teicoic acids. The cell wall is more susceptible to mechanical damage. With these bacteria there are further layers around the muerin consisting of lipid material. These are layers of polysaccharide, a layer of phospholipids, and next to the muerin is a layer of phophslipid. These act as a barrier to substances passing from the outside into the cell, such as penicillin, and also dyes, which is why these bacteria do not take up the Gramm stain. These layers also provide resistance to phagocytosis, therefore the bacteria are resistant to lysozymes. The cell wall is a good target for antimicrobial drugs. Lysozymes attack the polysacchyaride ‘backbone’ of the muerin, completely destroying the cell wall in Gramm positive bacteria. In Gramm negative bacteria the lipid-constituent layer

remains unharmed, but the muerin layer will be destroyed. However, the bacterial cell will remain alive. Some bacteria can survive without a cell wall. The mycoplasma is the smallest known bacteria to survive outside living cells, and they do not have a constant shape, i.e. they are pleomorphic. The capsule surrounds the cell wall, and usually is comprised of polysaccarhide, and is loosely attached to the cell wall. There is a direct correlation between the possession of a capsule and virulence of the organisms, e.g. Diplococcus pneumoniae will produce pneumonia if it has it’s capsule, but without the capsule the bacteria is less likely to produce the disease. The capsule also protects the bacteria from being phagocytised. Within the bacteria there is no nucleus, mitochondria, endoplastic reticulum, lysosomes or membranous structures. There is a single strand of DNA, forming a circular chromosome, and some ribosomes are also present. Bacterial motility. The usual structures to aid motility are the flagellae, which are sited on the outside of the cell membrane. They are not found on cocci, but are on most bacilli and some spirilla. They are usually formed from a protein material. They can be arranged in a number of ways : Monotrichous Amphitrichous Lophotrichous Peritrichous A pili is a structure used for adhesion found on the outside of some bacteria. They are almost are exclusive to Gramm negative organisms, and appear much shorter than flagellae. Genetic material can be exchanged between bacteria at the site of the pili. Endospores. These are round or oval bodies, formed inside the bacterial cell in adverse conditions to help the bacteria survive. They are not reproductive structures, but a mechanism which aids survival. Endospores may remain viable for many years through adverse conditions, but conditions improve the endospore will germinate and produce a new bacterial cell. Spores can be found in soil dust and air. Spores are formed mainly by the bacilli-type organisms, particularly those of the Bacillus and Clostridium groups. The Bacillus group are responsible for anthrax and tetanus, whilst the Clostridium group are responsible for botulism. Bacillus stearothermophilis is a heat resistant spore that we can use in a clinical situation. The spores are put into an autoclave, and after the cycle is complete they are allowed to germinate. If the autoclave is working correctly, then all spores, including this one, will be killed. If the autoclave does not reach the correct temperature, then this spore will survive the raised temperature, and will germinate successfully. This then indicates that the autoclave is faulty.

Bacterial growth. Individual bacteria at maximum size will split by binary fission into two bacteria. The maximum size of a bacteria is the size at which maximum efficiency occurs (it grows in volume but the increase in surface area is at a much slower rate, therefore the cell eventually becomes inefficient). The conditions required for bacterial growth are: 1. suitable nutrients 2. suitable atmosphere 3. suitable temperature 4. suitable pH 5. suitable osmotic potential 6. mosture. Bacteria require a readily available supply of organic nutrients, in particular oxygen, carbon, hydrogen, nitrogen, phosphorous, and sulphur. Essential minerals indlcude calcium, iron, potassium and magnesium, together with zinc, copper, cobalt, and magnesium. Different atmospheres will also benefit different organisms : • Obligate aerobes – these carry out aerobic respiration and therefore require oxygen from the atmosphere. They cannot survive the absence of oxygen. • Strict anaerobes – unable to grow and reproduce in the presence of oxygen, and usually die if oxygen is present. In areas where there is no oxygen present (i.e. devitalised tissues) these organisms can flourish, commonly producing gangrene in areas of damaged tissue. • Facultative anaerobes – can grow with or without the presence of oxygen. These are sub-divided into two groups – those that respire anaerobically whether oxygen is present or not, and those that respire anaerobically in the absence of oxygen and aerobically if oxygen is present. • Microaerophilic – obligate aerobes which only require a tiny amount of oxygen. If oxygen levels are too high, they will be destroyed.

Controlling the growth of micro-organisms. Microbial growth is controlled by physical and chemical means. Heat and autoclaves represent the physical method, whilst disinfectants represent the chemical method. The terms ‘sterilisation’, ‘disinfection’, ‘antisepsis’, and ‘germicide’ all refer to the destruction of micro-organisms. Sterilisation is the process of destroying all forms of microbial life, including spores. It is absolute, and there are no degrees of sterilisation. Disinfection is the process of destroying vegetative pathogens, but not necessarily endospores or viruses. Disinfectants tend to reduce or inhibit microbial growth, but normally do not sterilise. Antisepsis refers to chemical disinfection of the skin, mucous membranes, or other living tissues. A germicide is a chemical agent that kills microbes rapidly, but not necessarily their spores. The terms ‘bacteriostasis’ and ‘asepsis’ refer to the suppression of microbial growth. Bacteriostasis is a condition in which bacterial growth and multiplication are inhibited, but not killed. If removed, growth will resume. Asepsis is the absence of

pathogens from an object or area. Aseptic technique is designed to prevent the entry of pathogens into the body. Basic principles of controlling microbial growth. These apply to all methods of control of microbial growth : 1. agent used must be able to affect the micro-organisms directly; 2. the item must be cleaned so as to remove extraneous soil; 3. moisture is essential for the action of chemical agents; 4. killing of micro-organisms is not instantaneous, and the time required depends upon : a. the nature of the organism b. the nature of the agent used c. the numbers of organisms present d. the temperature. Gramm positive organisms are more susceptible to disinfectants and antiseptics than Gramm negative organisms. Pseudomonads are resistant to chemical agents, and will actively grow in some disinfectants and antiseptics. They can also survive in simple saline solutions, and are resistant to many antibiotics. They are a major cause of problems in hospitals as they are opportunistic pathogens in the absence of the normal flora of the body, i.e. when the normal flora has been suppressed during abti-biotic therapy. Micro-organisms are also more susceptible to chemical agents during their growth, but with an increase in age comes an increase in resistance. Endospores are much more resistant than vegetative cells, e.g. spores of C. Botulinum will survive after 5½ hours of boiling. Heat is more effective under acidic conditions, as are some chemical agents. Anti-microbial agents work by altering the permeability of the cell membrane, causing leakage of the contents and affecting growth. Other agents work by damaging the proteins or nucleic acids within the cell. If these proteins are destroyed then the cell is unable to synthesise proteins, leading to cell death. Physical methods of destruction. Heat. This is rapid and penetrates objects as well as clumps of micro-organisms, and all types of organism can be destroyed. It can be used in either a dry or moist form. In it’s dry form is kills by the oxidation effect, denaturing the cell’s proteins. In it’s moist form because the presence of the water allows the hydrogen bonds in the proteins to be broken down, denaturing the protein by a different method. With moist heat spores and bacteria are killed more rapidly at a given temperature for two reasons – chemically the proteins are denatured due to the breaking of the hydrogen bonds, whilst physically hot water has a greater heat content than air at the same temperature. Moist heat methods include boiling and autoclaving. Dry heat methods include flaming, incineration, and hot air sterilisation. Flaming is 100% effective, and is often used to needles or forceps, but it has limited use

otherwise. Incineration rapidly kills micro-organisms and is reliable. The use of hot ovens is not as efficient as moist heat, as higher temperatures are required for longer periods of time to ensure cell death, e.g. 120oC of dry heat will take 8 hours to achieve sterilisation. This is reduced to 20 minutes at 180oC. Sterilisation by this method is also effective for enclosed containers, and also items made from glass or metal, but not cloth or rubber. Filtration. This is the passing of a gas or liquid through a filter with pores small enough to prevent bacteria passing through. Viruses and larger proteins can also be removed. This method is used for liquids which would be destroyed by heat, i.e. antibiotics, and also for air-filtration. Radiation. Gamma rays will penetrate cells, killing them by destroying their DNA. This method is used especially for scalpel blade sterilisation. Ultra-violet light may also be used, especially for vaccine sterilisation, but this works very slowly. Chemical methods of destruction. Very few chemical agents actually achieve sterility, but they do reduce the microbial population to safe levels, destroying pathogens. Chemical agents should be selected so as to kill the organisms as quickly as possible. The following table illustrates some of the commonly used chemical agents. Agent Action Uses. Phenol. Disrupts the plasma No longer widely used due to its membrane and denatures irritating properties, and may also be proteins. teratogenic. It’s effects are also cumulative. It is the standard for measuring the effectiveness of other disinfectants (phenol co-efficient). Phenolics Disrupts the plasma These are derivatives of phenol and membrane and denatures are reactive even in the presence of proteins. organic material. Used to disinfect surfaces and instruments as well as skin surfaces and mucous membranes. Examples include cresol, Lysol, thymol, xylenol. Halogens. Iodine – inhibits protein These agents may act alone or as a function and is a strong component of inorganic/organic oxidiser compounds. They are both effective Chlorine – forms antiseptics. hypochlorous acid, a strong oxidiser which alters cellular components. Alcohols. Dehydrates and is a lipid Bacvtericidal and fungicidal, but solvent, acting on the cell ineffective against endospores and membrane. non-enveloped viruses. Used for skin swabbing and also wiping instruments.

Heavy Denature proteins metals and their compounds. Dyes. Interfere with cellular oxidation. Quarternary Inhibit enzymes, denature ammonium proteins and disrupt plasma compounds membranes. (QAC)

Acids and alkalis.

Cause hydrolysis and denaturing of proteins.

Aldehydes.

Inactivate proteins.

Oxidising agents.

Oxidise cellular components of cells.

Germicidal and antiseptic in action.

Antiseptic and antifungal. Surface-active agents. Bactericidal, bacteriostatic, fungicidal, and virucidal against enveloped viruses. They are colourless, odourless, tasteless, non-toxic, stable and easily diluted. Used for skin antisepsis and washing/disinfecting instruments, utensils, and rubber goods. Used on the skin to control dermatophytes, and as food preservatives to inhibit mould growths. Very effective agents. Used to preserve and embalm specimens. Will also inactivate micro-organisms and vaccines. Common example id formalin. Ozone can be used to replace chlorine for disinfecting water. Hydrogen peroxide is used as an antiseptic in deep wounds.

The normal flora of the body. Many organisms inhabit the body without causing us any harm, and is some cases are positively beneficial and essential for health. Mutuals are bacteria which actually aid some process in the body, such as vitamin K-producing bacteria in the gut. Commensals benefit from living on the body, but we gain no benefit directly from their presence. These mostly live on the skin. The areas on the body which mainly have normal flora are those areas which have an exterior connection, i.e. mouth. Those that have no exterior connection have no flora. The normal flora will utilise any nutrients that are available, preventing their use by pathogens. They also maintain certain physiological conditions within the body, such as pH, making it impossible for pathogens to flourish. They can also produce secretions which inhibit invading micro-organisms. Flora on the epithelial layers occupy specific attachment sites, and once these attachment sites are occuplied there is no-where for pathogens to attach and establish themselves. The major constituent of the normal flora is S. albus, making up around 90% of the population. Another common organism is S. aureus, found commonly in the nose and anus. Diphtheroids occur cutaneously, and commonly inhabit oily areas such as the

sebaceous glands. Streptococci are seldom found in normal flora, but α-haemolytic streptococci exist in the mouth and can spread to the skin. Gram negative bacilli form a very small proportion of the flora, and tend to occur in moist areas only as they cannot survive in dry areas. Nail flora – this is generally similar to that of skin, but in addition particles of dust and other materials can be trapped which contain spores and bacteria, eg, aspergillus, penicillium. Oral and upper respiratory tract – pharynx and trachea contain many potentially pathogenic organisms, and this is often the site of the initial colonisation of pathogens. Intestinal tract – the concentration of organisms increase towards the end of the small intestine. There is a high concentration in the colon, a high percentage of which are anaerobic (more than 90%). Urogenital tract – depends upon age, pH, and hormone levels. There are certain conditions in which normal flora may cause infection : • when taking antibiotics that are not specific to one type of bacteria, thereby killing all bacteria. This destroys the normal flora and the other organisms, such as fungi and yeasts, will multiply, causing infection, e.g. thrush. • Extreme and prolonged fatigue . • Debilitated patients, especially those recovering from major surgery, cancer, diabetes or chest conditions. • The use of drugs such as steroids, and possible drugs of abuse. • Large amounts of alcohol. • Mechanical damage to the skin or tissues. • Transmission of normal flora to another region. Microbial invasiveness and disease production. Disease production depends upon a number of factors : 1. host/microbial factors – a. portal on entry – the organisms enters the wrong portal it cannot cause disease. b. actual species – only certain micro-organisms cause disease. c. numbers of micro-organisms – salmonella poisoning requires over 1 million organisms, whereas only 1 rickittsia will cause Scrub Typhus. d. ability to multiply. e. must attach to epithelial surfaces. f. needs correct metabolic and nutritional situation. 2. specific microbial factors – a. production of a capsule for protection b. toxin production – endotoxins are part of the bacterial cell wall which is released on lysis or destruction. They are associated with Gramm negative bacteria and tend to be non-specific. They result in headache,

c. d. e. f. g. h. i.

fever, and nausea. Exotoxins are secreted into the surrounding environment and are very powerful. They are mainly associated with Gramm positive bacteria, and it is the exotoxin which causes the disease, e.g. clostridium tetani produces a toxin which results in Lockjaw. haemolysins – released by bacteria to break down red blood cells leukocidins – released by bacteria to destroy leukocytes coagulase – produced by some staphylococci to promote blood clotting fibrinolysis – produced by streptococci and breaks down a blood clot by destroying the fibrin hyaluronidase – breaks down hyaluronic acid, enabling bacteria to get in between the host’s cells collagenase – breaks down collagen in connective tissues penicillinase – breaks down penicillin and other similar substances.

Summary of factors necessary for disease to occur : The correct species must enter the right portal in sufficient numbers for that species to cause disease. They must have the ability to attach to epithelial surfaces, have the correct nutrients, temperature and environmental conditions for growth, and produce substances such as capsules, toxins and enzymes necessary for their survival and growth. States of bacterial infection. State of infection Subclinical (apparent) Dormant (latent) Accidental Opportunistic

Primary

Secondary Mixed Acute Chronic Localised Generalised

Description No detectable clinical symptoms of infection Carrier state Zoonosis and environmental exposure Infection caused by normal flora or transient bacteria when normal host defences compromised Clinically apparent invasion and multiplication of microbes in body tissues causing local tissue injury Microbial invasion subsequent to primary infection Two or more microbes infecting the same tissue Rapid onset, brief duration Prolonged duration Confined to a small area or an organ Disseminated to many

Examples Asymptomatic gonorrhea in women and men Typhoid carrier Anthrax Candida infection

Shigella, dystentery

Bacterial pneumonia, viral lung infections Anaerobic abscess Diphtheria Tuberculosis, leprosy Staphylococcal boil Gramm negative

Pyogenic Retrograde Fulminant

body regions Pus forming Microbes ascending a duct or tube against the flow of secretions or excretions Infections that occur suddenly and intensely

bacteraemia Streptococcal infection E.coli urinary tract infection Pneumonic plague