key: cord-022399-66mzbynu
authors: Hopkins, Graham; Pearson, Richard
title: Basic microbiology
date: 2009-05-15
journal: Ophthalmic Drugs
DOI: 10.1016/b978-0-7506-8864-2.50005-2
sha: 
doc_id: 22399
cord_uid: 66mzbynu

nan

• patients with 'red eye' • contact lens solutions and the claims made for them by manufacturers • preventive measures following contact tonometry and foreign body removal • the constituents of eye drops and the maintenance of sterility.

The science of microbiology covers organisms invisible to the naked eye. Microorganisms include protozoa, fungi, bacteria, rickettsia, chlamydia and viruses.

Protozoa and fungi are the only microorganisms that have eukaryotic cells similar in structure to those of higher organisms. Such cells have inclusions like nuclei and an endoplasmic reticulum. Fungi and protozoa can be either parasitic or free-living.

Bacteria are simpler cells (prokaryotic cells) but some species are capable of an independent existence in simple environments. However, many bacteria are parasitic or saprophytic and live on the tissues of living or dead organisms. Rickettsia and chlamydiae lack many of the structures found in more complicated cells and are thus obligate intracellular parasites.

Viruses are the simplest microorganisms and can multiply only by utilizing a host cell's biochemical systems.

Of the above, the bacteria have received most attention.

Bacteria are important because of their ubiquity -that is, their ability to infect and multiply in varied environments -and the ability of many types of bacteria to cause disease -their pathogenicity. To reduce problems caused by bacteria, it is important to understand something of their structure, growth, environmental and metabolic requirements, classification, relationship with disease and the particular problems they can cause to the eye.

The cytoplasm of bacterial (prokaryotic) cells ( Fig. 3.1) is notable for the absence of the discrete structures normally found in eukaryotic cells ( Fig. 3 enzymes are instead located on the cell membrane. There is also no endoplasmic reticulum or Golgi apparatus; the ribosomes are found free in the cytoplasm. There is no nucleus and no nuclear membrane, and when the cell divides there is no mitosis. Genetic material is carried on a single strand of DNA, unlike the genetic material of eukaryotic cells. which is organized into chromosomes. Some bacteria contain additional DNA molecules called plasmids, which are not essential for the organism's life but can confer on it very special properties such as resistance to antibiotics. Plasmids are most commonly found in Gramnegative bacteria and can spread from one cell to another. Surrounding the bacterial cell cytoplasm is a thin, selectively permeable, elastic, lipoprotein membrane -the plasma membrane. It is the site of action of many of the bacterial enzymes and controls entry of substances into the cell. Being elastic, the cell membrane does not determine the shape of the bacterial cell. This is the function of the cell wall, a rigid, permeable structure principally composed of a substance called murein. Because of the osmotic pressure of the cytoplasm, the cell membrane is usually pushed hard against the inside of the cell wall by a pressure of up to 20 atmospheres. The cell wall is relatively thick, especially in Gram-positive bacteria, and is chemically unrelated to the cell walls of higher plants.

In some bacteria, the cell wall is surrounded by a layer of extracellular material referred to as the capsule (if it is closely associated with the cell wall) or slime layer (if the relationship is looser). This is a poorly organized layer of large molecules such as polysaccharides or polypeptides. The effect of this layer is to impede the ingress of substances (useful and harmful) into the cell wall. The result is that the cells tend to grow and divide at a slower rate but are more resistant to antibacterial chemicals, viruses (bacteriophages), phagocytes and other adverse agents. This layer might also inhibit antibody formation against the bacteria, thereby rendering the bacteria more harmful to the body. Additionally, it can act to form an adhesion between the bacterial cell and a host cell, which also increases its virulence. One of the best known bacteria to form a capsule is Mycobacterium tuberculosum, the causative organism of tuberculosis.

Flagella are found on the outside of some types of bacteria; the number of flagella per cell is constant for each species. Flagella are long, filamentous structures containing a contractile protein, flagellin, which is similar to muscle myosin. The presence of flagella normally confers the ability for motion that, it is assumed, allows the bacterium to migrate to more favourable environments.

Some bacteria have the ability to move without possessing flagella. These are the spiral forms, which move by twisting the whole body.

Also projecting from the surface of some bacteria are fimbriae, formed of protein, which instead of facilitating motion act to hold the bacterial cell to a host cell or another bacterial cell. The fimbriae are very specific for the molecule to which they attach and fimbriated bacteria are found to be more virulent than those that lack these appendages.

Bacteria are ubiquitous and can exist in many environments that are far too hostile for the cells of higher organisms. More fastidious bacteria have requirements closer to those of the internal environment of animals and hence are more likely to be parasitic and pathogenic.

Nutritional All organisms have a requirement for carbon, hydrogen, oxygen and requirements nitrogen. As hydrogen and oxygen can be obtained from water, it is the requirement for the other elements -nitrogen and carbon -that is most critical. Some bacterial species can obtain their nutrient requirements from inorganic sources. They obtain energy from other sources, e.g. bacterial chlorophyll allows some bacteria to use sunlight as a source of energy and synthesize simple organic compounds. Others can utilize inorganic nitrogen, providing they are supplied with an organic source of carbon. Such organisms are found in soil and are responsible for maintaining its fertility. Yet others require both organic carbon and nitrogen to survive. Pathogenic bacteria need other complicated growth factors and minerals.

Oxygen requirements Although oxygen can be obtained from water, some types of bacteria need atmospheric oxygen and cannot exist without it. These bacteria are termed obligate aerobes. Others are the exact opposite and cannot exist in the presence of oxygen, requiring anaerobic situations (obligate anaerobes). The majority, however, are facultative anaerobes, which means they can exist in either the absence or presence of oxygen.

Physical conditions Different bacteria can exist at both high and low extremes of both pH and temperature. Pathogens prefer the medium state of pH 7 and 37°C. Acidophilic bacteria prefer a low pH, whereas basophiles like a high one. Thermophilic bacteria grow best at between 55 and 80°C, whereas the spores of Bacillus stearothermophilus can withstand boiling. Some organisms can exist at very low temperatures.

Growth Bacteria reproduce by binary fission. The cell divides and two equal daughter cells are formed. As there is no nucleus there is no mitosis. The time between a daughter cell being formed and itself dividing to form two new cells is called the generation time and varies greatly between species. It also varies with environmental conditions and the supply of nutrients. Some bacteria multiply very quickly and divide every 20 minutes. Others, like M. tuberculosus, take hours or even days.

When a new sterile environment with finite limits is colonized, the bacterial cell population goes through four phases (Fig. 3.3 ).

• Lag phase: the original innoculum remains dormant and no increase in number is seen.

• Log phase: there is an exponential growth in the number of organisms and the logarithm of the number of cells is directly proportional to time. It is during the early stages of this phase that the bacterial population is most susceptible to antibacterial agents.

• Stationary phase: the number of viable organisms remains constant because the number of new organisms is equal to the number dying. This phase can be brought about by a depletion of essential nutrients, a change in the oxygen level or an accumulation of metabolites which regulate the growth.

• Decline phase: the number of viable organisms declines. Sporulation Certain bacteria can produce endospores. These compact masses, which have a very resistant coat, are formed inside the vegetative cell. Once formed, the rest of the cell disintegrates, releasing the spore. Spores can withstand adverse environments that would be lethal to the vegetative cell. When the conditions are right, the spore germinates into one vegetative cell (sporulation is not a form of multiplication).

Once a pure colony of an organism has been isolated by successive culturing, it is often necessary to find out which organism is present. Not only genus and species require elucidation but also the particular strain. To elicit this information the following techniques can be used:

• microscopy and staining • differential media and biochemical tests • serological testing • bacteriophage typing. Microscopy and Gram's stain divides bacteria into Gram-positive and Gram-negative differential staining bacteria. Bacteria are fixed onto a microscopy slide and stained with a dark purple stain. The slide is then covered with an iodine solution that acts as a mordant, i.e. fixes the stain onto the organisms. The next step is the decolourizing process, in which the slide is treated with a solvent. A counterstain completes the process and the slide is viewed under the microscope. If the organism has resisted decolourization it is termed Gram positive and will appear purple under the microscope. If the original stain has been lost, the colour of the counterstain will show through and the organism will be deemed Gram negative. This is a fundamental method of classifying bacteria.

Other differential stains have been used. For example, acid-fast staining, in which the organisms are subjected to a decolourizing process using acid. Specific stains can be used to show the presence of sporeforming bacteria.

Examination under the microscope not only gives information about the organisms' staining characteristics but, of course, about the shape. Basically, bacteria can be spherical (cocci), rod-shaped (bacilli) or spiral. Cocci can be divided according to their form of aggregation. Some bacteria appear in just one direction and form chains (streptococci) whereas others give the appearance of a bunch of grapes (staphylococci). However, the appearance of aggregations under the microscope can sometimes be deceptive and other tests are necessary to differentiate between streptococci and staphylococci.

Differential media and Special media that support the growth of some types of bacteria and biochemical tests not others can be useful in bacterial typing. Other tests examine the organism's ability to break down hydrogen peroxide, to liquefy protein and to ferment certain sugars. Media containing blood are useful in differentiating streptococci.

Serological testing Bacteria possess many potentially antigenic substances and one of the body's defences against bacterial invasion is to produce antibodies to these antigens. These antibodies are specific to the antigens and this specificity can assist in the determination not only of the genus and species but also the strain of bacteria present.

Bacteriophage typing Bacteriophages are viruses that attack bacteria. They invade the bacterial cell just like any other host cell. Once inside they combine with the bacterial DNA and change the genetic material. This effect can be destructive and the whole cell is taken over, producing new phage particles. Bacteriophages are species specific to the bacterium they invade.

Viruses ( Fig. 3 .4) are much smaller than bacteria (18-300 nm). Whereas all known bacteria will be trapped by a 0.22 μm filter (sterilizing filter), many viruses will pass through, hence the term 'filtrable viruses'. Viruses can infect any form of higher organism and are usually divided into animal viruses, plant viruses and bacteriophages. Viruses consist of either RNA or DNA (never both) surrounded by a layer of protein (capsid) or a membrane (referred to as the envelope). The outer covering of the virus particle plays a vital role in the initial infection of the cell and the spread of a virus within the host. The components of the covering layer are very antigenic and are involved in the host's immune response. Capsids are rigid structures and tend to be very protective against adverse environments such as desiccation and detergents. Such viruses tend to retain their infectivity on fomites and will withstand the adverse conditions in the gut, i.e. low pH and the presence of proteases. Envelopes are less protective and can be disrupted by acids, detergents and drying out. Thus these virus particles must remain in aqueous solution to survive and must be transmitted by droplet infection, blood or other body fluids; they will not survive the gut.

The nucleic acid may be single strand or double strand. Viruses contain few if any enzymes and are entirely reliant on the host cell to bring about replication; they are obligate intracellular parasites. They vary greatly in size and in the number of genes they carry (from three to several hundred). 

Virus reproduction does not take place by binary fission but, generally, by the following pattern:

• The virus particle (virion) becomes attached to the surface of the cell.

Specific receptors are usually involved, which results in viral preference for certain cells within the host. For example, the AIDS (HIV) virus binds to CD4 receptors, which are found on T cells.

• The virus particle passes into the cell.

• The virus particle becomes uncoated, releasing the viral genetic material into the host cytoplasm if it is RNA. The genetic material of DNA viruses must be delivered into the nucleus.

• The viral genetic material induces the cell to produce different macromolecules that are essential for the production of new viral particles.

• Assembly of new virus particles takes place within the host cell; these are then released. The release can bring about disruption of the host cell. The new virus particles are available to infect new cells.

The classification of viruses is more difficult than that of bacteria. Many criteria are used in classifying viruses.

• morphology • nucleic acid type • immunological properties • transmission methods • host and cell tropisms • symptomatology and pathology.

The following are the main groups of animal viruses: 

These organisms are more complex than viruses but less complex than bacteria. They can multiply only in susceptible cells and, unlike viruses, contain both DNA and RNA. They multiply by binary fission and are susceptible to certain antibiotics. Chlamydiae exist in two forms: (1) as an elementary body (300 nm) that can exist outside the host body and is the infectious unit; and (2) as a reticulate body (1000 nm), which exists only inside the host cell and is not infectious.

Chlamydiae attack mucous membranes and inhibit host cell protein synthesis. They rely on the host to provide ATP, which they cannot generate. They synthesize their own nucleic acids and proteins. There are two species, Chlamydia trachomatis (which causes trachoma, inclusion conjunctivitis) and Chlamydia psittaci (which causes psittacosis).

As causative organisms of disease, fungi are less important than bacteria and viruses. Of the tens of thousands of species of fungus, probably only about 100 are pathogenic. Some of these are capable of producing very severe infections (e.g. filamentous keratitis), others result in more trivial infections (e.g. athlete's foot). Fungi can colonize non-living structures and lead to spoilation, e.g. hydrogel contact lenses. Fungi are composed of eukaryotic cells with the normal inclusions, e.g. a nucleus and mitochondria. They can be divided into four groups according to their structure:

• Moulds: these grow as a mycelium, which is composed of filamentous multicellular structures called hyphae. The mycelium is divided into the vegetative mycelium, which grows into the substrate and assimilates nutrients, and the aerial mycelium, which produces the spores either asexually by budding or sexually by the fusion of two cells.

• Yeasts: these fungi occur as single cells and reproduce by budding. • Yeast-like fungi: both hyphae and yeast cells exist together. • Dimorphic fungi: these fungi exist either as yeasts or as filaments. If they are grown on artificial media they appear as hyphae; when they inhabit a living host they occur as yeast cells.

Protozoal parasites are best known for their ability to colonize and, in some cases, parasitize the alimentary tract, particularly in hosts in tropical countries. However, protozoal parasites are capable of living in other parts of the body and of causing serious pathological conditions. The most important of these are the various forms of amoebae, which are primitive acellular organisms with a simple life cycle. They exist in two forms:

• the active trophozite, which moves around its environment using pseudopodia (the cytoplasm extends in one direction and the rest of the organism is drawn along to follow it)

• the dormant cyst, which -like the spores of bacteria -is more resistant than the active trophozite to unfavourable environments.

Reproduction is either by simple binary fission of the active trophozite or the formation of multinucleated cysts.

Some amoebae are free living whereas others, such as Entamoeba coli and Entamoeba gingivalis, inhabit parts of the alimentary tract as commensals. Others, such as Entamoeba hystolytica (the causative organism of ameobic dysentery), are potent pathogens. Naegleria fowleri causes a rapidly fatal (4 to 5 days) meningoencephalitis. Acanthamoeba is an important pathogen in causing keratitis, especially in wearers of hydrogel contact lenses (see Chapter 16).

Humans play host to a large number of bacteria that normally do no harm and, to varying degrees, contribute to the body's well-being. For example, bacteria live on the dead surface of the skin and prevent other more potentially dangerous organisms from occupying the site. Bacteria that inhabit the gut provide the body with the vitamin K that is essential for the production of prothrombin, in exchange for nutrients. These commensal organisms only maintain this mutually advantageous relationship providing they remain in their proper place. One tissue's commensal is another tissue's pathogen.

It is not always the microorganism itself that causes the harm, but substances that the organism produces. Some bacteria live and multiply in food, producing toxins as they do so. When the food is ingested, the toxins produce adverse effects. Clostridium botulinum is such an organism and the toxin it produces, botulinum toxin, is fatal in minute quantities. Staphylococcus aureus can also bring about food intoxication. Most microorganisms, however, cause disease by acting as parasites on the body, gaining access by a variety of routes:

• Direct contact: this normally means sexual contact; the disease is classed as a venereal disease. This method of transmission favours the very fastidious bacteria that exist only with difficulty outside the human body. Treponema pallidum (which causes syphilis) is so fastidious that it has never been grown on lifeless media.

• Indirect transmission: infection is passed from one person to another by an inanimate object (called a fomite), e.g. bedclothes, used dressings.

• Dust-borne infection: dust contains discarded human cells and dried water droplets. These are likely to carry bacteria, especially spores.

• Droplet infection: bacteria are present in the fine spray that is exhaled with forced expirations such as coughing and sneezing.

• Water-borne infection: water is an excellent medium for transmitting infection. Public health and sanitation prevent this until some disaster interrupts the supply of clean drinking water.

• Insect-borne infection: biting and sucking insects can carry organisms from an infected host and transfer them to a new one.

• Maternal transmission: infections can be passed from mother to child, either while the child is still in the womb or during birth (e.g. ophthalmia neonatorum).

The eye is at risk from infection by opportunistic and invasive organisms via a variety of routes. In addition to congenital ocular infections, microorganisms can gain access as a result of:

• direct contact, e.g. herpes simplex • airborne infections • insect-borne infections, e.g. trachoma • migration of bacteria from the nasopharynx • metastatic infection from other loci in the body • trauma, especially penetrating injuries • infected contact lenses • infected eyedrops and lotions • infected instruments.

The lids, cornea and conjunctiva, being the most exposed parts of the eye, are at most risk from infection. Infections of these tissues are far more common than those of deeper tissues but they are also less serious.

The eye is covered with tears, which contain a number of antimicrobial agents to reduce the incidence of infections. The result is a fairly low level of microbial contamination in the fornices. Tears contain immunoglobulins A, G and M (IgA, IgG and IgM; Reim 1983) in different proportions to those found in plasma, suggesting that their origin is secretory rather than just the result of filtration.

Tears contain two other agents with marked antibacterial properties: lysozyme and beta-lysin. Lysozyme is an enzyme capable of dissolving the cell wall of bacteria, especially Gram-positive bacteria. The level of lysozyme decreases with age and is reduced in patients with dry eye syndrome (Mackie & Seal 1976) . Beta-lysin acts principally on the cell membrane (Ford et al 1976) and works in concert with lysozyme. As the cell membrane is the site of action of the bacterial enzymes, the effect is quite marked. Beta-lysin is also present in aqueous humour.

Staphylococci Some staphylococci are normal inhabitants of the skin and mucous membranes, whereas other species are capable of producing conditions such as boils, abscesses and even a fatal septicaemia. Others can cause food poisoning by the liberation of an enterotoxin. Resistance to certain antibiotics develops easily; the term 'hospital Staph' used to be applied to some resistant forms -the modern term is MRSA (methicillin-resistant Staphylococcus aureus).

Staphylococci are differentiated from streptococci by the presence of an enzyme that breaks down hydrogen peroxide (catalase). Pathogenic staphylococci possess coagulase, which clots blood plasma.

In the eye, staphylococci can cause infections of the lids, lacrimal apparatus, conjunctiva and cornea (Davis et al 1978) . Infections of the lash follicle can result in the formation of a stye (hordeolum). Staphylococci can also produce acute or chronic blepharitis, which is sometimes associated with acute conjunctivitis (Brook 1980; Brook et al 1979; Brown 1978) . Staphylococci were also found to be present in a large number of cases of ophthalmia neonatorum in one study (Jarvis et al 1987) . The routine use of prophylactic eye ointments to prevent ophthalmia neonatorum has led to the emergence of resistant strains. Hedberg et al (1990) reported an outbreak of erythromycin-resistant staphylococcal conjunctivitis in a newborn nursery in which erythromycin eye ointment was used as a prophylactic agent. Following septicaemia, staphylococci have caused endophthalmitis (Bloomfield et al 1978) .

Because Staph. aureus is so common, it is often employed in the efficiency testing of preservative systems. Staphylococcus epidermis is normally considered to be a commensal and is a normal inhabitant of the skin. Unlike Staph. aureus, it produces white colonies. Maske et al (1986) found a higher than normal incidence in a group of patients with bacterial corneal ulcers. It has been suggested that Staph. epidermis releases a toxin to cause some of the signs of blepharitis and keratopathy (McGill et al 1982) .

Streptococci Streptococci lack the enzyme catalase and are characterized by their ability to cause haemolysis Complete haemolysis is brought about by beta-haemolytic streptococci; the haemolysis produced by alphahaemolytic species is incomplete and leads to the formation of a green pigment. There are also non-haemolytic streptococci.

Streptococci can produce local and general infections. One of the most common local infections of beta-haemolytic streptococci is the streptococcal sore throat, which in young children can extend into the middle ear to cause otitis media. On the skin they can cause impetigo. Betahaemolytic streptococci infections give rise to puerperal fever, wound sepsis and endocarditis. It is fortunate that penicillin continues to be effective against many strains of streptococci.

In the eye, streptococci can cause conjunctivitis, dacryoadenitis, dacryocystitis and blepharitis (Brook 1980) . Jones et al (1988) reported corneal ulcers, endophthalmitis, conjunctivitis and dacryocystitis resulting from streptococcal infections. The ability of streptococci to cause sightthreatening infections is of concern because many strains are not susceptible to gentamicin, an antibiotic often chosen to treat such infections.

The Neisseriae is a group of Gram-negative bacteria that include the normal flora of the respiratory system and the pathogens which cause meningitis (Neisseria meningitidis), and gonorrhoea (Neisseria gonorrhoeae).

In the eye, Neisseriae species can infect the lids, lacrimal apparatus and conjunctiva and N. gonorrhoea is best known as the one-time principal cause of ophthalmia neonatorum, an infection that occurs as the infant passes down the birth canal. The disease becomes manifest between the second and fifth day after birth, when the lids become swollen and there is a bilateral purulent discharge. The lids are tightly closed and difficult to open and the acute phase lasts for 4-6 weeks. The condition is treated with topical and systemic antibacterials. If treatment is not carried out, the cornea can become involved and the eye lost. However, other causative organisms, such as Chlamydia species, and other causes (paradoxically, the overenthusiastic use of silver nitrate) are more important today (Jarvis et al 1987) .

Corynebacterium Corynebacteria are non-motile Gram-positive rods; they do not form diphtheriae spores. Some species are normally resident in the human respiratory tract. Corynebacterium diphtheriae, when infected with the appropriate bacteriophage, produces a powerful exotoxin that causes diphtheria. This disease results in the growth of a membrane across the throat, leading to suffocation. It can similarly affect the eyelids, with the appearance of such membranes on the inner surface of the lids. The conjunctiva can become involved in the same way. Diphtheroids have been isolated in a proportion of infected conjunctivae (Brooke et al 1979; Brown 1978 ).

Clostridium species The Clostridia are a group of obligate anaerobes notorious for their pathogenicity. In particular, they include Clostridium botulinum, which, when it infects food, produces botulinum toxin. Although botulinum

toxin ingestion is potentially fatal, this substance has been used to paralyse the antagonist muscles in cases of paralytic strabismus (Elston & Lee 1985) and other ocular disorders (Alpar 1987 ). Clostridium tetani is a possible infectant of deep wounds and prophylaxis against the effects of its toxin is routine. Other Clostridia, such as Clostridium perfringens, Clostridium welchii and Clostridium oedematiens cause gangrene. Gas gangrene of the lids has been reported (Crock et al 1985) .

This is by far the biggest group of pathogens, most of which are facultative anaerobes.

Pseudomonas Pseudomonas aeruginosa is perhaps the most notorious of bacteria for aeruginosa causing ocular problems and is normally found in small numbers in the gut and on the skin. It is a common contaminant of water and has been cultured from jacuzzis (Brett 1985) . Its numbers are kept in check by the presence of other organisms but as it is resistant to many antibiotics it can gain dominance if the surrounding organisms are suppressed.

Ps. aeruginosa produces a bluish green colour when grown on media. It has a characteristic odour and is pyogenic, the presence of green pus suggesting the presence of a pseudomonal infection. Ps. aeruginosa is an opportunistic organism and is normally kept at bay by the body's defence mechanisms. If these are breached, a serious infection often results. Ps. aeruginosa can infect burns, especially if of large area, and can also gain hold in patients who are immunecompromised. Ps. aeruginosa is an extremely versatile organism in that it can metabolize fluorescein and hydroxybenzoates as carbon sources for energy, which means that it can survive in conditions that are alien to most other organisms. The organism is susceptible to antibiotics such as gentamicin and polymixin.

In the eye, Ps. aeruginosa can produce meibomitis, conjunctivitis and corneal ulcers and is one of the causes of ophthalmia neonatorum (Cole et al 1980) . Should access be gained to the sterile interior of the eye, then panophthalmitis might result and, indeed, has been responsible for causing more than one serious case of hospital acquired disease leading to the loss of an eye (Crompton 1978) . Ps. aeruginosa is an important test organism for contact lens solutions and eyedrop preservative systems, not only for its virulence when an infection is established but also because of its biochemical versatility, which sometimes makes it difficult to eradicate.

Haemophilus species These small, aerobic organisms get their name from their requirement for enriched media containing blood for culturing in vitro. They include certain important human pathogenic organisms. Haemophilus influenzae is a secondary invader, which helps to produce some of the symptoms of influenza and can produce inflammation in most parts of the respiratory tract. Bordetella pertussis is another member of this group that affects the respiratory system, causing whooping cough, which is transmitted by airborne infections from one person to another. B. pertussis cannot exist for long periods outside the body. Similarly, Haemophilus ducreyi is so fastidious in its requirements that it can only be transmitted sexually and, consequently, is the causative organism of chancroid, a form of venereal disease.

H. influenzae and H. ducreyi can infect ocular tissues. Two members of this group, however, are particularly noted for their ability to cause conjunctivitis: Haemophilus aegyptius (Haemophilus conjunctivitidis, Koch-Weeks bacillus) is often the cause of acute epidemic conjunctivitis, especially in school children and Moraxella lacunata (Morax-Axenfeld bacillus) is another well-known causative organism of conjunctivitis.

The most important members of the herpes group as far as the eye is concerned are herpes zoster, herpes simplex and cytomegalovirus.

Herpes zoster (varicella) virus results in chickenpox in children. This is a mild, highly contagious disease characterized by a vesicular rash. The disease leaves the patient with a continuing immunity to the disease. In the adult, a reactivation of the virus leads to shingles, in which an area of the body becomes covered with a painful rash. Evidently the virus is stored in a sensory ganglion, the shingles attack being caused by a migration of the virus along the nerve root. When the nerve affected is the ophthalmic division of the trigeminal nerve, the area served by it exhibits signs, i.e. the eye, the orbit and surrounding areas. This is known as herpes zoster ophthalmicus, in which the cornea becomes inflamed and oedematous, and sensitivity may be impaired permanently. Secondary infection can occur, leading to ulceration and scarring.

Herpes simplex can be differentiated into types I and II. Type II is associated with genital herpes and neonatal herpes. Transplacental infection with type II virus has led to the development of neonatal cataract (Cibis & Burge 1971) . Type I causes cold sores, inflammation of the oral cavity, encephalitis and dendritic ulcers.

Dendritic ulcers are so called because of their branching pattern. As the ulcer extends it might lose this appearance and become amoeboid or geographic. The patient complains of pain, photophobia, blurring of vision and a watery discharge (unlike that from bacterial conjunctivitis). In the early stages, infection affects only the epithelium, later progressing to the superficial stroma. The cornea becomes oedematous and there is further loss of stroma and possible vascularization. The condition is treated with intense local antiviral therapy. Herpes simplex can also produce a keratoconjunctivitis similar to that caused by adenovirus 8 (Darougar et al 1978) (see below).

The third virus in this group is cytomegalovirus, which normally inhabits the female reproductive tract giving rise to congenital infections. Congenital infections can give rise to chorioretinitis, optic atrophy and cataract.

Adenovirus 8 Adenovirus 8 gives rise to epidemic keratoconjunctivitis sometimes called 'eye hospital eye' because of its possible transmission by contaminated instruments. The infection produces severe acute conjunctivitis, which can spread, leading to keratitis. Marked discomfort can last for months. Adenovirus was the cause of 8% of cases of acute conjunctivitis in one study (Wishart et al 1984) .

Pox viruses This group of viruses includes smallpox and cowpox. A relatively uncommon skin condition affecting young adults and childrenmolluscum contagiosum -is caused by a pox virus. Transparent nodules (2-3 mm in diameter) appear on the skin of the arm, legs, back and face, with possible involvement of the lid margins and conjunctiva. This condition is sometimes seen in patients with AIDS and AIDS-related complex (ARC).

The best known member of this group is rubella (German measles), which can be passed from mother to baby in the uterus, leading to congenital defects in 30% of the children of mothers suffering rubella in the first trimester of pregnancy. Particularly affected are the heart, ears and eyes. Ophthalmic defects lead to microphthalmia, cataracts and congenital glaucoma.

Retroviruses The human immunodeficiency virus (HIV) is present in many of the body fluids of affected individuals, including tears. Although there has been no recorded case of transmission via infected contact lenses, it has become a point of concern of contact lens practitioners. The virus has also been recovered from contact lenses worn by patients with AIDS and ARC (AIDS-related complex; Tervo et al 1986) . AIDS can have certain ocular manifestations, partially because the patients are, from the very nature of the disease, more likely to develop opportunistic infections such as cytomegalovirus retinitis. Conjunctival Kaposi's sarcoma is another ocular complication (Kanski 1987) .

This dimorphic opportunistic fungus is normally found in the mucous membranes of the mouth, vagina, gut and eye (Liotet et al 1980) . It causes oral thrush in newborn infants and terminally ill patients. In the eye it can cause corneal ulcers, conjunctivitis and severe uveitis.

Aspergillus niger This fungus, which is not dimorphic, grows in the form of mycelia. Often found in vegetable matter, it can cause bronchial problems; it can also produce severe local infection in the eye, especially after injudicious use of local corticosteroids, which tend to mask the clinical signs of the infection, allowing it to get a stronger foothold. A case of Aspergillus panophthalmitis has been reported in a patient after excision of a pterygium who received beta radiation treatment (Margo et al 1988) . Aspergillus niger, which also can be found in the healthy eye (Liotet et al 1980) , can infect hydrogel contact lenses and destroy them. Other species of Aspergillus have been implicated in contact lens contamination. For example, Yamaguchi (1984) reported growth on a contact lens of Aspergillus flavus and Filppi et al (1973) found that Aspergillus fumagatus penetrated hydrogel contact lenses. Aspergillus species are not the only ones to infect contact lenses. Yamamoto et al (1979) found Cephalosporium acremonium growing on a contact lens worn for the treatment of metaherpetic keratitis.

trachomatis) endemic in many areas. Where it is endemic, it affects over 90% of the population. Associated with poor living conditions, this organism is passed on by insects and contaminated objects such as bedclothes (fomites). It is sometimes a resident of the female genital tract (Barton et al 1985) and can produce a form of ophthalmia neonatorum (Markham 1979) . The incidence of chlamydial ophthalmia neonatorum varies. In one study in America, 1.4% of all newborn babies acquired chlamydial conjunctivitis (Schacter et al 1979) and similar findings were reported in Sweden (Persson et al 1983) and Wolverhampton, UK (Preece et al 1989) .

The last report concluded that screening for chlamydial antigen was not justified as the condition could be easily treated with oral erythromycin. The condition starts as a mild inflammation of the conjunctiva, with the development of small follicles, which become larger. The cornea is invaded and vascularized, resulting in pannus, which can lead to severe scarring and contraction, which causes deformity of the eyelids. Symblepharon and trichiasis are also seen. In temperate climates, the organism results in inclusion conjunctivitis.

Acanthamoebae like Naegleria can infect the brain, producing a form of amoebic encephalitis (Ma et al 1990) . Amoebic keratitis has been reported as a result of infection with Acanthamoebae species (Moore et al 1986) . The patients in the report were myopes corrected with hydrogel contact lenses who had used salt tablets dissolved in distilled water during disinfection procedures. This infection has so far proved to be difficult to treat.

Many 'agents' have the ability to kill or inactivate microorganisms. Within this broad term we encompass the body's defence mechanisms, i.e. the white blood cells and circulating antibodies of the blood, the

gastric hydrochloric acid and the lyzosyme and beta-lysin of tears; other microorganisms, such as bacteriophages, must also be considered as antimicrobial agents.

Here we are concerned with three basic groups:

• physical agents capable of rendering objects and chemicals free of contamination • antimicrobial preservatives that are incorporated into solutions to maintain sterility • chemotherapeutic agents used either to treat or prevent an infection in the body.

It is important to define certain terms that are relevant to this subject and are sometimes used incorrectly:

• Sterilization: the killing or removal of all viable organisms (including bacterial spores) from an object or pharmaceutical product by the use of chemical or physical agents.

• Disinfection: a lesser process than sterilization by which the capacity of an object to cause infection is removed. A disinfected product might not be sterile.

• Antisepsis: a similar degree of decontamination as disinfection but referring to solutions and chemicals that are safe to apply to surfaces of the body.

• Chemotherapeutic agents: described as bactericidal or bacteriostatic;

the former are actually capable of killing the bacteria (although not necessarily bacterial spores) whereas bacteriostatic agents prevent bacteria from growing and rely on the body's own defence mechanism to get rid of the organisms.

All physical agents are forms of energy and the antimicrobial action is dependent on supplying sufficient to cause disruption to the cell. Bacteria and other microorganisms are far more resistant to adverse situations than animal cells and can withstand environments that would be quickly lethal to us. The effect of antimicrobial agents follows a firstorder reaction in which the log number of survivors is inversely proportional to the time. The time for 1 log cycle reduction, i.e. the time taken for 90% of the bacteria to be killed, is called the D value or decimal death time, this value reducing as the antimicrobial effect of the antimicrobial increases.

Heat Heat is one of the best-known disinfecting and sterilizing agents. It is used for sterilizing solutions (providing that the substances are thermostable), dressings and some instruments. The effectiveness of heat is increased by the presence of water, especially if the pH is raised, the use of moist heat bringing hydrolysis to bear on the organisms as well as pyrolysis. Temperatures of around 60°C will kill most viruses, as well as the vegetative cells of pathogenic bacteria and fungi, whereas boiling brings about the demise of spores of pathogenic bacteria. However, there are organisms whose spores will withstand boiling for long periods. Therefore, to obtain sterility without compromising the product, autoclaves are used. These work on the 'pressure cooker' principle by heating the product in steam (not air) to a defined temperature and specified time, which is usually 121°C for 15 minutes. The use of dry heat is far less efficient and temperatures of up to 160°C for 1 hour are needed to kill spores. Disinfection, as opposed to sterilization, can be brought about by boiling for 10-15 minutes.

Temperatures below boiling can reduce the number of microorganisms present and are used for materials that cannot withstand heating at high temperatures; milk is pasteurized at 60-70°C, for example. The temperature inside a soft lens storage subjected to heating by steam will not reach boiling but the temperature attained (95°C) is very bactericidal. Thermal disinfection of contact lenses is discussed in Chapter 11.

Freezing Freezing cultures of bacteria will markedly reduce the number of bacteria, as a proportion will be damaged by the formation of ice crystals. However, the rest will survive in a dormant state even at temperatures as low as that of liquid nitrogen. Indeed, this process is used to store cultures of bacteria.

Ionizing radiation This technique is used for disposable plastic items and for paper products such as fluorescein-impregnated strips. All types of radiation are lethal to microorganisms (alpha, beta and gamma rays). Usually it is gamma rays that are used at a dosage of 2.5-3.5 Mrad.

Ultraviolet radiation Light is only bactericidal at low wavelengths (240-280 nm -the UVC region) and at this level does not penetrate well, making it suitable only for surface and aerial disinfection.

Filtration Solutions of thermolabile drugs can be sterilized by passing the solution through a 0.22 μm filter, which retains all bacteria (the smallest bacterium is about 0-5 μm). The filters, which are sterilized before use, will not remove viral contamination. Ultrasonics Sound will kill bacteria but high power inputs are required. Ultrasonic cleaners with antibacterial cleaners have been used on contact lenses (see Chapter 11).

A whole range of substances is incorporated into products to prevent the growth of microorganisms. These are used in foods and drinks and cosmetics but are most important in multidose sterile pharmaceutical solutions to ensure that the product is protected from microbial attack while it is in use.

These compounds are selected for their ability to kill or inhibit the growth of microorganisms, particularly bacteria and fungi. The rate of kill represented by the D value depends on the concentration of the preservative, but is not always a simple inverse relationship, i.e. with the D value inversely proportional to the concentration of the preservative compound. With some compounds the effect is exponential, with a reduction of concentration to half of the original leading to an increase in D value of a factor of 2 8 or 256. Such compounds are thus quickly inactivated by dilution.

These agents can damage human cells and it is necessary to use them in as low a concentration as possible to reduce toxicity, the final concentration therefore representing a compromise between safety and efficacy. To achieve greater efficacy without increasing the toxic effects, mixtures of preservatives are often used. Many such agents have been used in the past; the following are those in common use.

Benzalkonium chloride Benzalkonium chloride (BAK) has a detergent action that disrupts the cell membrane; it is by far the most commonly used preservative for eye drops. Benzalkonium chloride has a disruptive effect on the tear film when used in concentrations of 0.01% and greater (Wilson et al 1975) . It is often found combined with ethylene diamine tetra-acetic acid sodium edentate (EDTA), which is a chelating agent that combines with divalent ions (normally calcium) to form a non-ionizable complex. EDTA has a slight antibacterial action of its own but is principally used to enhance the bactericidal action of benzalkonium chloride.

They produce mercury ions that react with sulphydryl groups of essential enzymes. They are slower in action against certain organisms but are less quickly inactivated by dilution than other compounds. Unlike benzalkonium chloride, mercury compounds are not potentiated by the addition of EDTA (Richards & Reary 1972) . In fact, Morton (1985) found that EDTA actually reduces the antimicrobial efficacy of thiomersal. Significant penetration of mercury-containing compounds into the aqueous humour following their use has been recorded by Winder et al (1980) . These compounds have been demonstrated to have cytotoxic effects that are time and concentration dependent (Takahashi 1982) but that are less than those of benzalkonium chloride (Gasset et al 1974) , and their use in many contact lens solutions has led to the increasing incidence of allergic reactions (Gold 1983 ).

Chlorhexidine gluconate Chlorhexidine is a useful alternative to benzalkonium chloride and is used when the latter is incompatible with the active ingredient. It is very toxic to the corneal endothelium in concentrations of 20 μg/mL and, if the epithelium is perfused, the result is a sloughing of the cells without corneal swelling (Green et al 1980) .

Oxidizing agents Strong oxidizing agents are very bactericidal. Probably the best known is hydrogen peroxide, which kills most vegetative forms at a concentration of 3-6%; stronger concentrations ( > 10%) will dispose of spores. The halogens are also strong oxidizing agents and part of the action of iodine-and chlorine-based disinfectant systems is the oxidation of essential enzymes. Although iodine in simple solution is still occasionally used it is most often complexed with another compound to form an iodophor. Iodine can be complexed with poly-vinylpyrrolidine to form povidone iodine.

Other compounds Other compounds that have been used include chlorbutol, cetrimide, phenylethanol, hydroxybenzoates and chlorocresol.

The treatment of infections has evolved somewhat since the treatment of syphilis with mercurial compounds. Developments have led to the introduction of agents that are more effective against the infecting organism and less toxic to the host.

Anti-infectives tend to be specific against groups of organisms, e.g. antibacterials and antifungals, although there is some overlap. Certain antibacterials are effective against chlamydiae. The mode of action of antibacterial agents varies greatly. To produce their desired effect without producing a toxic reaction from the patient they must interfere with some specific function important to the parasitizing cells. These include:

Inhibition of the Many of the common antibiotics produce their activity by interfering formation of with the formation of cell walls. Penicillin and the other beta-lactam the cell wall antibiotics, such as the cephalosporins, work in this manner, as do vancomycin and bacitracin. The antibiotic molecules combine with enzymes responsible for the synthesis of the cell wall, preventing new wall being laid down and the destruction of existing material. In the normal bacterial cell the membrane is pushed against the cell wall by osmotic pressure. Without this constraint water balance is not maintained and cell death occurs. These antibiotics are most effective against populations which are actively growing as this is the stage of maximum cell wall production. Such bacteria are normally bactericidal.

The cell membrane of the prokaryotic cell is even more important than the cell membrane that of the eukaryotic cell because the former lacks mitochondria and so respiratory membranes are located on the membrane. Any disruption of the membrane, as well as interfering with the transport of substances into and out of the cell, will have an effect on cell respiration.

Inhibition of protein Many of the more modern antibiotic groups can be found under this synthesis heading -the tetracyclines, aminoglycosides, macrolides chloramphenicol and clindamycin. Their site of action is the ribosomes, where they either bind directly or prevent the binding of tRNA. Many of these antibiotics are bacteriostatic.

The most important group in this section are the quinolones, of which acid synthesis new members are constantly introduced. Also included are rifampicin, used in the modern treatment of tuberculosis, and metronidazole, often used in the dental treatment of anaerobic infections. Rifampicin inhibits the formation of RNA whereas the quinolones interfere the development of DNA.

The best known antimetabolites are the sulphonamides, which interfere with uptake of para-amino-benzoic acid (PABA), which prevents the synthesis of folic acid. Trimethroprim is taken up by an enzyme necessary for the ultilization of folic acid. Interference with the metabolism of folic acid inhibits the production of new genetic material. The administration of antimicrobial agents for the treatment of infection requires the achievement of adequate levels of the antimicrobial agent as quickly as possible. For many antibiotics, this level is referred to as the minimum inhibitory concentration (MIC), which is the concentration that prevents visible growth after a 24-hour incubation period. Unless the route of administration can achieve a level substantially higher than the MIC for the invading organism, it is unlikely that successful treatment of the infection will be achieved. For bactericidal agents, the figure quoted is minimum bactericidal concentration (MBC), which is the concentration that kills 99.99% of the bacterial cells.

The higher the concentration of the agent, the greater will be the effect on the organism. Failure to achieve these levels can lead to the development of resistant strains. In any population there will be a proportion of organisms that are resistant to the agent. In the presence of the agent these will be selected and will form the majority of the population. Some bacteria develop resistance by altering their metabolic pathways to avoid those with which the antimicrobial interacts. Other bacteria produce enzymes capable of destroying the chemical, e.g. penicillinase, which is an enzyme produced by certain strains of staphylococci and is capable of breaking down penicillin.

Although there has been no recorded case of a patient contracting AIDS from a contaminated contact lens, this condition has highlighted the necessity for good practice hygiene. The AIDS virus is not the only organism (opportunist or invasive) that could be transmitted in an optometrist's practice and simple disinfection procedures should be employed in order to protect both practitioner and patient.

The first consideration is one of cleanliness, as clean objects and surfaces are easier to disinfect and will remain uncontaminated for longer. Normal contaminants will harbour bacteria, protect them from antibacterial agents, provide them with nutrients and inactivate disinfectants. Jacobs (1986) has laid down simple infection control guidelines for optometrists and contact lens practitioners. Basically, anything that can be boiled without adversely affecting its performance should be, e.g. bowls, soft contact lenses. Items of equipment that will touch the eye should be swabbed with 70% alcohol, e.g. tonometer heads, chin rests, trial frames. Working surfaces should be treated with 1% sodium hypochlorite solution, which is effective against bacteria and viruses. At levels as low as 500 ppm (0.05%) this solution destroys herpes simplex, adenovirus 8 and enterovirus 70 within 10 minutes (Naginton et al 1983) . Such procedures will protect the patient more than will a prophylactic eye drop.

In the interests of self-protection, the practitioner should have no open cut uncovered but the added precaution of wearing gloves is only necessary for high-risk patients, e.g. patients who are HIV positive.

Botulinum toxin and its uses in the treatment of ocular disorders

Detection of Chlamydia trachomatis in the vaginal vault of women who have had hysterectomies

Endophthalmitis following staphylococcal sepsis in renal failure patients

Pseudomonas aeruginosa and whirlpools

Anaerobic and aerobic bacterial flora of acute conjunctivitis in children

Anaerobic and aerobic bacteriology of acute conjunctivitis

The conjunctival flora of nursing home patients and staff

Herpes simplex virus induced cataracts

Pseudomonas ophthalmia neonatorum: a cause of blindness

Gas gangrene infection of the eye and orbit

Medical ethics and hospital-acquired disease

Acute follicular conjunctivitis and keratoconjunctivitis References due to Herpes simplex virus in London

Paralytic strabismus: the role of botulinum toxin

Penetration of hydrophilic contact lenses by Aspergillus fumagatus

Identification of a nonlysozymal bacteriocidal factor (betalysin) in human tears and aqueous humor

Cytotoxicity of ophthalmic preservatives

The war on thiomersal

Chlorhexidine effects on corneal epithelium and endothelium

Outbreak of erythromycin resistant staphylococcal conjunctivitis in a new born nursery

Infection control guidelines for optometrists and contact lens practitioners

Asbell P A 1987 Ophthalmia neonatorum. Study of a decade of experience at the Mount Sinai Hospital

Ocular manifestation of AIDS

Conjunctival flora of healthy people

Quantitative tear lysozyme essay in units of activity per microlitre

Aspergillus panophthalmitis complicating treatment of pterygium

Genital tract to eye infection -tissue culture of Chlamydia trachomatis

Management of bacterial corneal ulcers

Pathophysiology of bacterial infection in the external eye

Radial keratoneuritis as a presenting sign in acanthamoeba keratins

EDTA reduces the antimicrobial efficacy of thiomersal

Tonometer disinfection and viruses

Neonatal chlamydial eye infection: an epidemiological and clinical study

Chlamydia trachomatis infection in infants. A prospective study

Normal and pathological components of tears and conjunctival secretion

Changes in antibacterial activity of thiomersal and PMN on autoclaving with certain adjuvants

Prospective study of chlamydial infection in neonates

Cytotoxicity of mercurial preservatives in cell culture

Recovery of HTLV-III from contact lenses

Effect of benzalkonium chloride on the stability of the precorneal tear film in rabbit and man

Penetration of mercury from ophthalmic preservatives into the human eye

Prevalence of acute conjunctivitis caused by chlamydia adenovirus, and Herpes simplex virus in an ophthalmic casualty department

Herpetic keratitis and corneal destruction

Treacher Collins prize essay. Inflammatory diseases of the outer eye

Fungus growth on soft contact lenses with different water contents

Fungal invasion of a therapeutic soft contact lens and cornea