Ophthalmic Medical Microbiology PDF

MSc in Clinical Optometry: Principles of Therapeutics Unit 3: Part 2 OPHTHALMIC MEDICAL MICROBIOLOGY: MICROBIOLOGY OF ANTERIOR SEGMENT EYE CONDITIONS Description: A wide range of micro-organisms is known to cause ocular infectious disease. This part aims to summarise the process of microbial recover...

MSc in Clinical Optometry: Principles of Therapeutics Unit 3: Part 2 OPHTHALMIC MEDICAL MICROBIOLOGY: MICROBIOLOGY OF ANTERIOR SEGMENT EYE CONDITIONS Description: A wide range of micro-organisms is known to cause ocular infectious disease. This part aims to summarise the process of microbial recovery and identification from ocular samples. Hours: Eight Learning outcomes Following successful completion of this module you should be able to: Describe a routine laboratory protocol for the identification of micro-organisms Demonstrate knowledge of the normal ocular biota and describe the most common micro-organisms involved in anterior segment eye disease Describe the effect of disinfection/sterilisation on micro-organisms and the role of anti-infectives Introduction Many commensal (generally harmless) microbes are normally present at various sites in the body where they rarely cause an infection. It is important, therefore, to recognise these organisms in the laboratory, even though they have no clinical significance when compared with pathogenic microbes that are capable of causing clinical infections. For example, if a faecal swab from a patient with suspected food poisoning is sent to the laboratory, only the known pathogens such as Salmonella, Campylobacter, Shigella or Staphylococcus (Staph.) aureus, if isolated, are reported. The normal bacterial flora of the intestine would be identified as commensals and would, therefore, be of no clinical significance. In ophthalmic microbiology, as the eye is sited externally and structurally is a very delicate organ, the basic microbiological principles explained above do not always apply. The cornea is prone to infection from many types of bacteria, fungi and other organisms, which in other clinical infections would be given commensal status. OPHTHALMIC MEDICAL MICROBIOLOGY pathogenic micro-organisms, as is the case for other mucosa. Normal ocular biota There has been debate in the past as to whether the The most common micro-organisms isolated from the lid ocular surface is transiently colonised by micro- and conjunctiva are Gram-positive bacteria, specifically organisms arising from the skin or whether there is a coagulase negative Staphyolococci, Corynebacteria and resident microbiota, analogous to that of the oral Propionibacterium species. Other isolates include mucosa or gastrointestinal tract. Approximately half of Staph. aureus, Micrococcus, Bacillus and Bacteroides all ocular samples will recover micro-organisms and the species. Gram-negative micro-organisms are isolated in lids tend to harbour larger numbers of micro-organisms less than 5% of cases and they tend to be present as than the conjunctiva. The cornea and anterior chamber transient micro-organisms rather than persistent are considered to be sterile. It is generally accepted that colonisers in normal healthy eyes. At birth, the bacterial the external ocular surface is sparsely colonised and it spectrum differs slightly, with a preponderance of seems less likely that the low numbers of micro- Lactobacillus, Bifidobacterium, Corynebacterium, and organisms present do prevent colonisation by Peptococcus species, and coagulase negative Microbiology of anterior segment eye conditions Staphylococcus. By five days of age, the biota is similar to that of the adult, although during childhood there is a more frequent streptococcal colonisation. In adults, fungi are more rarely isolated, although regional variations have been reported, with fungal recovery from the eyelids occurring in 2-52% of samples and in 2-37% of conjunctival samples. Ocular biota are influenced by regional variations and by sleep. The number and frequency of recovery of Gram-positive micro-organisms increases with sleep such that patching of the eye overnight following foreign body removal should be accompanied by prophylactic antibiosis. Figure 1: The agar sandwich technique for the Obtaining a sample culture of micro-organisms adherent to a contact Microbial analysis of a corneal infection requires direct lens. This figure demonstrates thousands of scraping of the corneal ulcer to provide smears and colonies of Gram-negative bacteria that were cultures. In severe cases, a corneal biopsy may be adherent to the surface of the lens, and released taken and in vivo confocal microscopy has been used to into the media upon immersion into hot agar. assist in the diagnosis of Acanthamoeba and fungal disease. The bulbar conjunctiva, fornices and lids may be sampled with moistened cotton or calcium alginate causative organisms or molecular techniques such as swabs. Corneal scrapes are taken following instillation the polymerise chain reaction (PCR), where specific of unpreserved single use topical anaesthetics target sequences of microbial DNA are amplified. PCR Unpreserved anaesthetics are preferable to avoid the has particularly been used to establish a diagnosis of microbicidal effects of preservatives. The base and herpetic disease. Other molecular typing techniques leading edge of the ulcer are scraped using a platinum established for a range of micro-organisms include spatula or surgical blade and the material spread onto a Southern blotting, random amplification of polymorphic clean glass slide and air dried prior to staining for DNA (RAPD) and mitochondrial DNA fingerprinting by microscopy. Subsequent scrapes are inoculated onto restriction fragment length polymorphism (RFLP). solid or liquid media. Smears taken for microscopy can be stained according to the likely causative agent (see Routine laboratory protocol later). If the patient presents wearing contact lenses Direct microscopy is carried out on the samples and, if these are most often cultured using either the agar necessary, a report is given to the clinician immediately. sandwich technique (Figure 1) or lens maceration The appropriate bacterial culture media is inoculated technique. with the sample, and this is then incubated at 37°C (the temperature may vary according to the type of bacteria, Microbial growth on the various media is enumerated see section on culturing bacteria) for 24–48 hours, in after the appropriate incubation and the number of the correct atmospheric conditions. Culture plates and colony forming units (cfu) of each distinct colony type is tubes are examined after 24 and 48 hours of incubation. calculated. Representative bacterial colonies from each If significant bacterial growth is present, antibiotic plate are then Gram stained and identified employing sensitivity tests are performed and evaluated after a standard microbiological methods. It is usual to use further 24 hours. standard biochemical techniques or commercially available tests, such as API strips (Figure 2). Staining for microscopy Gram stain Laboratory diagnosis of viral infection may include direct The Gram stain enables microbiologists to make the examination of tissue scrapes, cell culture to isolate first major identification of all types of bacteria. It is Figure 2: API test kit 2 Microbiology of anterior segment eye conditions carried out as follows: Gaseous requirements - Fix material onto a microscope slide by heat or - Aerobic bacteria require a normal atmosphere. alcohol treatment - Anaerobic bacteria require there to be - Stain with crystal violet for two minutes absolutely no free oxygen present. - Wash off with water - Microaerophilic bacteria require an atmosphere - Treat with iodine solution for two minutes with a reduced oxygen level. - Wash off with water and decolourise rapidly with - Facultative anaerobes (e.g. Staphylococci) will acetone or alcohol for a maximum of five grow under aerobic and anaerobic conditions. seconds - Fastidious bacteria (e.g. N. gonorrhoeae or - Wash well meningitidis) require an additional 5% carbon - Counterstain with neutral red, safranine or dilute dioxide added for primary isolation from patient carbol fuchsin material. - Wash, dry and add coverslip - Add immersion oil to slide and examine under Fungal growth requirements 100x oil immersion objective Fungi are easy to grow (on bread after three days at kitchen temperature). The nutrients they require for The results of the stain can be interpreted as follows: growth are absorbed from the environment. Moisture is - Gram-positive bacteria stain blue essential for fungi to grow. - Gram-negative bacteria stain red - Yeast cells stain blue Bacteriological media - Fungal hyphae stain variably Laboratory bacteriological media must contain the - Connective tissue, cells and other materials following ingredients: stain red - Carbohydrates as a source of carbon Numerous other alternative staining methods are - Amino-acids (protein) as a source of nitrogen commonly used: - Salts, for example sodium, potassium, magnesium and calcium Giemsa stain - Water The Giemsa stain is used for cytological staining of - Agar as a setting agent tissue, cells, bacteria and fungi. A wide range of bacteriological media is available Zeil-Neelsen stain commercially. Enrichment media is used to encourage The Zeil-Neelsen stain is used for the demonstration of the growth of fastidious bacteria. Selective media is acid-fast bacteria. The Mycobacteria, which include the used for the detection of specific types of bacteria and tubercule bacilli, are acid-alcohol fast. to suppress the growth of commensals that may be present in the sample. Flourescent dyes Flourescent dyes (rhodamine and fluorescein) can be Blood agar used, sometimes with specific antibodies, to Blood agar is used most commonly in the medical demonstrate the presence of bacteria, fungi and laboratory. It contains all of the above ingredients, as viruses. Specimens are viewed under a fluorescent well as 7% horse or sheep blood. It will support most microscope. types of bacteria, although some important exceptions are the diphtheria and tubercule bacilli. Immuno-peroxidase Immuno-peroxidase methods are useful for Chocolate agar demonstrating the presence of Acanthamoeba species. Chocolate agar is blood agar that has been heated at Culturing micro-organisms 60°C for 10 minutes. It is used for the isolation of As discussed, the basic requirements for bacterial bacteria with fastidious growth requirements, i.e. growth are water, as vegetative bacteria are 95% water, Neisseria and Haemophilus species. and heat. The culture temperature used routinely in microbiology laboratories is 37°C, although this differs Sabouraud’s agar for some organisms; for example, psychrophiles are Sabouraud’s agar is used for the isolation of fungi. grown at a temperature of 15°C or less, mesophiles at 25–45°C, and thermophiles in excess of 45°C. Broth media There are many types of liquid media. An advantage of External ocular infections tend to be caused by broth media is that it will encourage the growth of small mesophilic micro-organisms, which may be aerobes, numbers of organisms. It will also dilute any residual facultative aerobes or microaerophilic. antibiotic that may be present in patient eye samples. 3 Microbiology of anterior segment eye conditions Bacterial growth ANTERIOR SEGMENT EYE DISEASE This can be divided into the following phases: A wide range of micro-organisms is known to cause - Lag phase ocular infectious disease. Table 1 summarises the - Logarithmic phase (rapid multiplication) common causative micro-organisms for different ocular - Stationary phase diseases. - Decline phase (death of cells) Bacteria generally reproduce by binary fission. Table 1: Common causative micro-organisms in ocular infections Ocular site Disease Micro-organisms Staph. aureus Coagulase-negative staphylococci Blepharitis Streptococcus species Lid margin Blepharoconjunctivitis Gram-negative rods Blepharokeratoconjunctivitis Corynebacterium species Propionibacterium acnes Staph. aureus Strep. pneumonaie Conjunctiva Acute bacterial conjunctivitis (adult) Haemophilus influenzae Proteus species Moraxella species Haemophilus influenzae Conjunctiva Acute bacterial conjunctivitis (childhood) Neisseria gonorrhoeae Staph. Aureus Mycobacterium tuberculosis Conjunctiva Phlyctenular conjunctivitis Staph. Aureus Strep. pyogenes Conjunctiva Membranous conjunctivitis Neisseria species Corynebacterium species Conjunctiva Inclusion conjunctivitis Chlamydia trachomatis Staph. aureus Coagulase negative staphylococci Strep. pnemomoniae Pseudomonas aeruginosa Other Gram-negative bacteria (e.g. Moraxella, Branhamella, Proteus and Serratia) Cornea Microbial keratitis Haemophilus influenza Nocardia species Chlamydia trachomatis Aspergillus niger Aspergillus fumigates Fusarium species Candida species 4 Microbiology of anterior segment eye conditions Ocular site Disease Micro-organisms Pseudomonas aeruginosa Serratia marcescens Other Gram-negative bacteria Cornea Contact lens-related microbial keratitis Staph. aureus Strep. pneumonia Acanthamoeba polyphaga Acanthamoeba castellani Corneo-scleral junction Marginal keratitis Staph. Aureus Actinomyces israelii Canaliculi Canaliculitis Bacteroides species Staph. aureus Anterior chamber Hypopyon Strep. pneumoniae Moraxella Sclera Scleritis Strep. pneumoniae Staph. aureus Pseudomonas species Strep. species Anterior chamber Endophthalmitis Enteric micro-organisms Coagulase negative staphylococci Propionibacterium Blepharitis Streptococcus species Blepharitis is a bacterial infection of the lids, which may The beta-haemolytic strains, Lancefield Group be an acute or, more frequently, a chronic condition. A, can cause severe blepharitis and fascitis. Patients with blepharitis may have a generalised Alpha-haemolytic strains are a part of the eczematous infection. Specimens are taken with cotton- normal skin flora and rarely cause lid infections wool swabs moistened with a bacteriological nutrient (see also Bacterial conjunctivitis). broth and cultured on blood agar plates. Moistened Gram-negative rods swabs, when wiped over the lid margins, pick up more Pseudomonas and Proteus species may cause organisms than dry swabs. Blood agar plates are inoculated with the swabs using a standard technique to blepharitis. isolate single bacterial colonies. In order of frequency of Corynebacterium species isolation, the following bacteria may cause blepharitis. There are many species of Corynebacteria and they are commensals of the skin. A few clinical Staphylococcus aureus reports implicate this species of bacteria in Staph. aureus are usually responsible for chronic lid disease (see also Bacterial clinical cases of bacterial blepharitis. They may conjunctivitis). cause lid abscesses (see also Bacterial conjunctivitis). Propionibacterium acnes Propionibacterium acnes may have a Coagulase-negative Staphylococci pathogenic role, but are accepted as a skin These Gram-positive cocci are the major commensal. commensals of the skin. There are over 20 different species of which S. epidermidis is the Acute bacterial conjunctivitis most common. Patients with chronic infections In acute bacterial conjunctivitis, the eye is usually muco- may have an allergic response to these and purulent. In order to identify the causative microbe, other skin commensals. Patients are given samples are taken from the conjunctival sac with a broad-spectrum antibiotic therapy to reduce the sterile swab or plastic disposable loupe. Ideally, the normal skin flora. sample is immediately ‘plated out’ onto blood and/or chocolate agar. In order of frequency of isolation, the following bacteria cause conjunctivitis: 5 Microbiology of anterior segment eye conditions Adult Haemophilus influenzae Haemophilus influenzae are Gram-negative, Staphylococcus aureus pyogenic cocco-bacilli that cause the ‘pink eye’ Staph. aureus are Gram-positive, coagulase- syndrome frequently seen in the Spring months. positive, pyogenic cocci. They cause styes that are usually found on the bottom lid. They Proteus species commonly cause wound infections, eczema, Pseudomonass aeruginosa and other Gram- boils, otitis and food poisoning. Some strains negative bacteria such as Proteus species may are resistant to many antibiotics and are known infrequently cause conjunctivitis. as MRSA (Methicillin Resistant Staph. aureus; Figures 3 and 4). Moraxella species Moraxella species are Gram-negative rods that Streptococcus (Strep.) pneumoniae classically cause angular conjunctivitis and are Strep. pneumoniae are Gram-positive, alpha- a commensal of the naso-pharynx (Figure 6). haemolytic, pyogenic cocci (Figure 5). Figure 5: Gram stain of a corneal scrape from a patient with MK showing S. Pneumonia Figure 3: Growth of S. Aureus on blood agar (Pneumococcus). Magnification x 1500. plate after 24 hours’ incubation at 37⁰C. Figure 4: Gram stain of a corneal scrape from a Figure 6: Corneal scrape form a patient with MK patient with MK showing Staph. Aureus. showing Gram-negative, large diplo-bacilli, Magnification x 1500. Moraxella osloensis. Magnification x 1500. 6 Microbiology of anterior segment eye conditions immunosuppressed are particularly vulnerable to MK. Children All patients who have severe MK should be admitted to In addition to Haemophilus influenza and Staph. aureus: hospital as in-patients and receive intense topical treatment using fortified antibiotics. Sub-conjunctival Neisseria gonorrhoeae and parenteral treatment may be given but is of limited Neisseria gonorrhoeae are Gram-negative cocci value. that are often seen intracellularly. They cause severe haemorrhagic muco-purulent 1. Bacterial MK conjunctivitis, often with corneal involvement. In order of frequency of isolation, the following bacteria This is a notifiable disease and neonates may cause MK: be born already infected with it (Figure 7). Staphylococcus aureus Staph. aureus has been isolated from over 50% of MK cases in the UK. Coagulase negative staphylococci There are over forty recognised species of coagulase negative staphylococci. Major entities including Staph. lugdunensis, Staph. haemolyticus. Streptococcus species Strep. pneumoniae cause a severe muco- purulent infection. ‘Viridans’ streptococci cause a crystalline keratopathy. The beta-haemolytic Streptococci are rarely isolated from cases of Figure 7: Smear of conjunctival pus from a MK. patient with severe muco-purulent haemorrhagic Pseudomonas aeruginosa conjunctivitis showing small, Gram-negative P. aeruginosa are frequently isolated from diplo-cocci, Neisseria gonorrhoeae. Note that the contact lens wearers. They can, within 48 bacteria are contained within the cytoplasm of hours, penetrate the full thickness of the four of the polymorphonuclear neutrophil white cornea, due to their invasiveness and blood cells. Magnification x 1500. production of endotoxins. Pseudomonas are found throughout the environment, particularly in water, and are naturally resistant to many chemical agents and antibiotics. Pseudomonas, Membranous conjunctivitis together with MRSA, are the most common In addition to Neisseria species: causes of nosocomial (hospital acquired) Streptococcus pyogenes infections (Figures 8 and 9). Beta-haemolytic Streptococci can cause fascitis and are very pyogenic. Corynebacterium xerosis Corynebacterium xerosis are Gram-positive rods seen in clusters that mimic Chinese lettering. They are THE commensal of the conjunctival sac and belong to the same species as the diphtheria bacillus; in fact, microscopically they look identical. Microbial keratitis Microbial keratitis (MK) is an infection of the cornea caused by bacteria, viruses (sometimes with a secondary bacterial infection), fungi, yeasts and amoeba. In fact, it is possible for almost any microbe to opportunistically infect the cornea. The infecting microbe usually gains access to the cornea when the epithelium is damaged due to trauma. The ability of Figure 8: Blood agar plate inoculated with 25 µl bacteria to adhere to damaged and normal epithelium, of contact lens solution from a patient’s storage thereby resisting the washing action of tears, enables case. A confluent growth of P. aeruginosa is them to cause infection. Children, the elderly, those with present. a history of previous infection and the 7 Microbiology of anterior segment eye conditions Fungal MK is rarely seen in the UK but is commonly found in humid warm climates in parts of the Americas, the Indian sub-continent and the Far East. Many different species of fungi have been reported in the ophthalmic literature as causing MK. Aspergillus niger and Aspergillus fumigatus These are widely found in the environment of the UK. They are found on all types of grasses, particularly in mouldy hay. As with all hyphal fungi, aerial spores are produced and these are disseminated into the environment by the wind. Although rare, cases of MK caused by both species have been reported. Fusarium species Figure 9: Corneal scrapings showing Gram Fusarium cause acute MK, during which the negative rods, P. aeruginosa. Magnification x cornea is severely scarred. Several cases have 1500. been reported in the UK but affected patients Moraxella species Moraxella are Gram-negative diplo-bacilli. Although of low pathogenicity elsewhere, Moraxella cause severe MK. They are very sensitive to a wide range of antibiotics but the corneal stroma is slow to heal following an infection. Branhamella species Branhamella are Gram-negative cocci similar to Neisseria, but of very low pathogenicity. A commensal of the respiratory tract, they are responsible for a few cases of MK. Proteus and Serratia species Figure 10: Fungal keratitis: the long filaments These Gram-negative rods have been the with Gram variable staining are Fusarium solani. causative organisms of MK. Only a few cases Magnification x 1500. have been reported, usually in immunocompromised patients. have usually been infected abroad (Figure 10). Haemophilus influenzae 3. MK caused by yeasts Haemophilus influenzae usually cause Yeasts are single round bodies, 4–6 microns in conjunctivitis, but have occasionally been diameter. They are Gram-positive, reproduce by isolated from the cornea. ‘budding’ and can produce pseudohyphae in tissue. Candida albicans is most frequently isolated from Nocardia species patients with MK but Candida paropsilosis has also Nocardia are Gram-positive rods. They are been found in several patients. The patient usually has pleomorphic, microaerophilic and prefer to grow a predisposing infection or is immunosuppressed. at 28°C. They are of low pathogenicity, but have Candida species must be treated with antifungals. been isolated from the cornea and in general medicine from immunosuppressed patients. 4. Amoebic MK Amoebae are free-living organisms found in water, mud Chlamydia trachomatis and water-cooling tanks. They were first identified in the Chlamydia trachomatis is Gram-indeterminate cornea in 1969. When introduced into the cornea they (i.e. cannot be stained with the Gram stain). invade the stroma down to Descemet’s membrane Structurally the organism is Gram-negative. within a few days. With the advent of contact lens wear, the number of acanthamoebic cases has risen 2. Fungal MK markedly. Amoeba feed on Gram-negative rods, which are found in large numbers in the lens storage cases of 8 Microbiology of anterior segment eye conditions non-compliant patients. When the solutions are not Escherichia coli (a Gram-negative rod). The plates are changed regularly, a biofilm forms and bacterial incubated at 37°C for three–six days. The surface of the multiplication occurs. The conditions in the case are agar is examined microscopically at 100 x then ideal for amoebae to grow. The active, vegetative magnification. If no growth is evident, they are re- incubated at 28°C for up to a further 10 days. Amoebae may be detected by this method at 36 hours but the average time is six days. Microscopically, many staining methods may be used to demonstrate the presence of amoebae in tissues. The best ones are the immuno-peroxidase and indirect flourescent methods, in which specific acanthamoebic anti-sera are used. Amoebal MK is treated with brolene (propamidine), neomycin, chlorhexidine, antifungals, and PHMB (polyhexamethyl biguanide). The cysts are very resistant to most drugs and may re-infect grafted material. Marginal keratitis Figure 11: Immuno-peroxidase staining of a Marginal keratitis can be caused by Staph. aureus histopathological section showing toxins and manifests itself as inflammation of the Acanthamoeba species; the brown bodies are corneoscleral junction. amoebae trophozoites. Canaliculitis This is usually a chronic condition and the patient may describe a history of having had epiphora over many months, if not years. As the canaliculi are totally blocked, anaerobic conditions exist within these ducts. For the most effective treatment, the ducts are syringed out and the patient given penicillin G. The following bacteria cause canaliculitis: Actinomyces (A.) israelii A. israelii are anaerobic, Gram-positive, filamentous organisms. They are found in the ‘cheesy’ concretion that may be expressed from the canaliculi (Figure 13). Bacteroides species Bacteroides are Gram-negative, filamentous Figure 12: Immunoflourescent staining of a section of cornea showing Acanthamoeba species; the trophozoites are the yellow bodies within the section. amoebae are called trophozoites, and in the non-active stage they are known as cysts (Figures 11 and 12). Amoebal MK is caused by Acanthamoeba polyphaga and Acanthamoeba castellani, of which the former is isolated most frequently. These species form pentagonal cysts, which are easily recognised microscopically. Ideally, two corneal biopsies are taken for laboratory investigation: one for histological examination and the other for culture. For culture, the corneal material is Figure 13: A smear of pus from a patient with carefully ground up and inoculated onto plain canaliculitis showing a Gram-positive clump of nonnutrient agar plates that have been seeded with filaments, which are anaerobic. 9 Microbiology of anterior segment eye conditions rods. They are usually found in conjunction with Actinomyces and are also strictly anaerobic. Hypopyon A hypopyon ulcer is caused by an infection of the anterior chamber of the eye and is usually bacterial. A sample of aqueous is collected and examined microscopically, and the bacteria cultured. Staph. aureus, Strep. pneumoniae and Moraxella species are the most frequently isolated organisms. Endophthalmitis This is usually caused by trauma or exogenous infection. Rarely, endophthalmitis is caused by endogenous infection, i.e. yeast infection in drug abusers and in AIDS or immunosuppressed patients. Samples of vitreous are collected directly into a 5-ml Figure 15: Gram stain of a smear of vitreous syringe and sent to the laboratory for bacteriological from a patient with chronic endophthalmitis, five examination. An aqueous sample is also sent in most weeks after cataract surgery, showing a large cases. Microscopical examination is performed clump of P. acnes. Magnification x 1500. immediately and the remaining vitreous is inoculated onto blood, chocolate and Sabouraud’s agar, and a variety of liquid media. In patients with a clinical history Sensitivity testing may be performed by tube dilution of trauma, including post-operative cases, Staph. techniques, using liquid media, or by the disc diffusion aureus, Pseudomonas, Streptococcus and method, which is currently the method of choice Enterobacterium and various fungi have all been reported (Figure 14). Chronic endophthalmitis Coagulase-negative Staphylococci and Propionibacterium are two types of bacteria that have been isolated post-operatively from the vitreous of several patients following cataract extraction and the insertion of plastic lens implants. When introduced into the eye, these skin commensals may adhere to the implant. The infection may not produce clinical symptoms for many weeks post-operatively (Figure 15). Treatment of anterior segment microbial disease There remains debate in the literature on the merits of culture guided versus empirical therapy. Armed with knowledge of the likely pathogen based on epidemiology, common causative organisms for the region and clinical Figure 14: Gram stain of a smear of vitreous manifestation, a broad spectrum antibiotic with activity from a patient with endophthalmitis showing against common causative agents is commonly Candida parapsilosis (yeast). Besides the yeast administered as initial empirical therapy. Upon cells, pseudohyphae are also present. presentation of cases of moderate, severe or Magnification x 1500. unresponsive disease, it is usual to attempt to isolate the causative micro-organism. However, it should be borne in mind that, in the case of MK, approximately 50% of cases worldwide (Figure 16). The zones of inhibition of may be culture negative. Isolation and culture aim to bacterial growth on the surface of a sensitivity agar identify the infecting micro-organism, determine antibiotic plate are compared with the zones produced by a sensitivities and aid in the administration of appropriate control organism. If some of the test organism’s zones therapy. of inhibition are markedly less than the control zones, resistance to those antibiotics is indicated (see later). After isolation, and either after or concurrent with Antibiotics that are effective against a wide range of identification of the infecting micro-organism, the bacteria (Gram-positive and Gram-negative cocci and antibiotic sensitivity of the infecting micro-organism is rods) are classified as broad spectrum, and those that tested. This is most commonly performed for bacteria as are usually only effective against specific micro- there are a range of antibiotics available and not as organisms are classified as narrow spectrum. Antibiotics commonly performed for fungi, viruses or are further classified into bacteriostatic, which inhibit Acanthamoeba. bacterial growth, and bacteriocidal, which inhibit and 10 Microbiology of anterior segment eye conditions choice for patients with bacterial blepharitis Antibiotic Mode of Use Fusidic acid Topical antibiotic - narrow spectrum, primarily for Staphylococci Gentamicin Topical aminoglycoside antibiotic used in treatment of external bacterial infections. Effective against Gram-negative bacteria including Pseudomonas Levofloxacin A topical broad spectrum quinolone antibiotic Neomycin A topical aminoglycoside broad spectrum antibiotic Figure 16: Demonstration of sensitivity and resistance to antimicrobials often uses disc Ofloxacin Systemic/topical quinolone diffusion methods. In this case, the bacterium is antibacterial agent used in the resistant to penicillin (p) but sensitive to treatment of superficial bacterial ciprofloxacin (C), gentamicin (G), tobramycin (T) infection. Effective against Gram- positive and Gram-negative and vanomycin (V). organisms including Pseudomonas Polymyxin B May also be used topically and destroy bacteria. Table 2 lists common systemic and sulphate subconjunctivally in the treatment of topical antibiotics and their mode of use. (no longer infections of the eye caused by commercially susceptible strains of Table 2: Examples of common systemic and topical available in the Pseudomonas antibiotics and their mode of use UK) Propamidine A topical aromatic diamidine Antibiotic Mode of Use isetionate/Diprom disinfectant effective against Gram- Amikasin Effective systemic treatment opropramidine positive, less so against Gram- against serious Gram-negative isetionate negative infections resistant to gentamicin Tetracycline Systemic antibacterial - popular Ampicillin Effective systemic treatment choice for patients with bacterial against serious Gram-negative blepharitis - it is useful against infections resistant to gentamicin Gram-positive and Gram-negative bacteria and some protozoa Bacitracin zinc Systemic antibacterial not often used, but resistance indicates Ticarcillin Systemic antibacterial effective pencilillinase production by against infections due to organism Pseudomonas and Proteus spp. Ceftazidime Topical antibacterial used in conjunction with Polymyxin B and neomycin and is effective against Treatment of microbial keratitis Gram-positive infections The main aims of current therapeutic strategies when treating MK is to eradicate the infectious agent with anti- Cefuroxime Systemic broad-spectrum antibiotic microbial compounds and to modulate the host immune Chloramphenicol Topical/systemic broad-spectrum response with corticosteroids to prevent scarring and antibiotic, a popular choice for potential vision impairment. The major advantage of conjunctivitis. Not suitable topical instillation of anti-microbial agents is that high for treatment of Pseudomonas concentrations of drug are applied at the site of infection infections and the toxic effects normally associated with systemic delivery are avoided. Initial management of MK requires Ciprofloxacin Systemic/topical quinolone intensive therapy, which, in the case of anti-bacterial antibacterial agent - broad therapy, usually involves instillation of antibiotic drops spectrum, for Gram-negative and every 30 minutes to one hour for up to 48 hours, and for staphylococcal infections longer periods, in the cases of mycotic and viral infection. Disease duration, severity and cost to treat is Erythromycin Systemic antibacterial - popular 11 Microbiology of anterior segment eye conditions strongly associated with the causative organism. been reported, although penetrating keratoplasty can be Systemic absorption of potentially toxic eye drops can necessary to recover useful vision. occur through the conjunctiva, the nasolacrimal system, the oropharynx, the digestive system, and the skin. It is Mechanisms of antibiotic/drug resistance estimated that as much as 80% of each eye drop Since the introduction of antimicrobials, there have been passes to the nasolacrimal system and is available for increasing reports of the spread of microbial resistance. absorption. We may already be at a stage where certain infections, e.g. infections with MRSA, vancomycin resistant Standard anti-bacterial therapy for MK usually involves Enterococci and multi-drug resistant Pseudomonas are monotherapy with fluoroquinolone eye drops. Fortified almost impossible to treat effectively. Also, certain antibiotics (aminoglycosides and cephalosporin) are viruses, especially HIV, have been rapidly developing reserved for the more severe cases of ulceration. In resistance to the new antivirals. One reason for the addition to those antibiotics/combinations mentioned, speed of resistance development is the spread of vancomycin can be used for Staphylococcus infections resistance genes between bacteria. whereas gentamicin is used for Gram-negative infections. The trend to not culture and undertake Bacteria can exchange genetic information via a variety antibiotic sensitivity testing of MK can lead to incorrect of mechanisms. Cells can harbour non-chromosomal treatment and ultimately poor visual outcome. genetic elements called plasmids. These commonly Tobramycin, an aminoglycoside, inhibits bacterial encode for resistance. Plasmids replicate autonomously protein synthesis. Fluoroquinolones, such as but can, on occasion, become integrated into the ciprofloxacin and ofloxacin, affect DNA synthesis by bacterial chromosomes. Plasmids can be transferred inhibiting the DNA gyrase enzyme of bacteria. This from one cell to another by a process called enzyme is essential for bacterial replication and, as conjugation. If a plasmid that can mediate its such, fluoroquinolones are bactericidal. Vancomycin is a conjugation becomes integrated into the chromosome it systemic antibiotic that inhibits the synthesis of bacterial has the potential, when released from the chromosome, cell walls. to pick up extra genetic material from the chromosome which can then be moved to other cells during Antivirals, antifungals, anti-amoebic and anti- conjugation. A second genetic element that can mediate parasitic treatments resistance to antibiotics is a transposon. Transposons Viral, fungal and amoebic infections are all extremely are moveable genetic elements that reside either within difficult to treat. Viral keratitis may require oral antiviral the chromosome or within a plasmid. Finally, antibiotic treatment for up to one year. Amoebic and fungal resistance can be mediated by direct mutagenesis of a keratitis require long duration intensive and aggressive chromosomal gene. Usually bacteria either modify the therapy. The most common class of anti-infective used target of the antibiotic, remove the antibiotic from their to treat viral infections includes aciclovir and similar cytoplasm by rapid efflux pumps or enzymatically drugs. Acyclovir acts to inhibit DNA polymerase modify the antibiotic. These steps may be simple or enzymes in Herpes viruses. The viral DNA polymerases more complicated involving up to ten different genes mistake aciclovir for the nucleotide thymidine and that need to be acquired to become resistant to a single incorporate aciclovir into viral DNA leading to chain antibiotic. termination and prevention of viral replication. Sterilisation and disinfection Fungal diseases are usually treated with application of Disinfection is designed to destroy harmful micro- azoles. Often anti-fungal treatment has to be prescribed organisms. Disinfectants are compounds that kill micro- in combination with debridement of the cornea to organisms and may or may not kill spores, but are not improve drug penetration. As fungal cells are more safe to apply to living tissues. Common disinfectants similar to human cells, many of the anti-fungal agents include chlorine compounds such as hypochlorites and are also toxic when administered systemically to human detergents such as quaternary ammonium compounds. patients. Anti-fungal therapy involves initial treatment Some disinfectants are powerful enough to eliminate all with a topical broad spectrum agent, and subsequent life forms from an area and are given the name sterilants. treatment with more specific topical anti-fungal drugs. Detergents disrupt cell membranes, and hypochlorites are strong oxidising agents. Acanthamoeba keratitis is often treated with multiple antibiotics including antifungals, and hospitalisation is Sterilisation is a procedure of making free from live often necessary. Medical management of micro-organisms (usually by heat or chemical means). Acanthamoeba keratitis is complicated by the resistance Sterilisation is the complete removal of all life forms of these micro-organisms to most of the commonly used from a given area (note that this includes viruses even agents. Successful treatment using ketoconazole, though they are not really living). Treatments causing miconazole, and propamidine isethionate (Brolene) has sterilisation tend to be drastic and can sometimes alter the chemistry of the object being treated. The most common process for sterilising an object is via autoclaving. This process involves the use of moist heat and pressure. Commonly, objects are subjected to water heated under pressure to 121°C. Most bacteria, 12 Microbiology of anterior segment eye conditions viruses, fungi and spores are inactivated. Inactivation the recommendations to deactivate prions include: for occurs as the heat destroys biological compounds such heat resistant materials, the use of sodium hydroxide in as DNA, the cell membrane causing lysis of cells and an autoclave cycle, followed by rinsing and re-autoclaving denaturation of critical enzymes. Prions are notoriously in a normal environment. Bleach (5% sodium hypochlorite stable to heat and may not be inactivated by the normal - 50000ppm) has been found to be one of the most autoclave temperatures. As autoclaving uses heat, effective agents against prions. The College of thermally unstable materials are unsuitable, however, Optometrists have produced guidance to the profession on other sterilising processes are available. These infection control (including disinfection protocols). These alternative processes include ethylene oxide vapour and are available from the College website. gamma irradiation. Ethylene oxide is an alkylating agent, i.e. it attaches C2H5 to biochemical groups Conclusion rendering them inactive. Gamma irradiation is an ionising In this the second part of Unit 3 of the Principles of radiation that causes disruptions in many biochemical Therapeutics module, the process of microbial recovery molecules. At very high levels gamma rays can denature and identification from ocular samples has been explored proteins, while at lower doses, gamma rays collide with and the range of micro-organisms known to cause ocular various molecules (often water), producing highly reactive infections has also been discussed. Consequences of species, such as hydroxyl and hydride radicals. Gram- invading micro-organisms include MK, bacterial negative bacteria are more sensitive to radiation than are conjunctivitis, canaliculitis, blepharitis, marginal keratitis, most Gram-positive bacteria. Spores of Bacillus and hypopyon and endophthalmitis. Clostridium are more resistant still, due to special proteins in the spore that are thought to protect the DNA. Molds tend to have similar resistance ranges to those of most Acknowledgements and further reading bacteria, and yeasts are a bit more resistant than bacteria to radiation. However, certain bacteria, e.g. Deinococcus Levinson, W.E. (2016). Review of medical microbiology th radiodurans, can withstand ten times the radiation that and immunology. 14 Edition. Lange kills Escherichia coli and this ability stems from its unusual th efficiency in repairing double-stranded breaks in the DNA. Microbiology 4 Edition. Eds Davis BD, Dulbecco R, Eisen A disadvantage is that this type of radiation degrades HN, Ginsberg HS. JB Lippincott, Philadelphia, PA, 1997 some plastic gels. Sharma, S. (2012). Diagnosis of infectious diseases of As mentioned above, not all micro-organisms are equally the eye. Eye 26: 177-84. sensitive to disinfection/sterilisation. The general order of susceptibility is: viruses with lipid membranes > vegetative Wilcox, M.D.P. (2003). Pathogenesis of infectious bacteria > fungi > non-lipid viruses > Mycobacteria > anterior segment disease. In: The anterior eye and bacterial spores > prions. Current sterilisation techniques therapeutics: diagnosis and management. are mostly ineffective in deactivating prions. Therefore, Ed. F.Stapleton. Butterworth Heinemann 13