Burn injuries are most often caused when the skin comes in direct contact with a naked flame or a hot surface. Scalding can occur when there is contact with a hot liquid. There are several other types of burns such as chemical, caused by strong acids or bases, and radiation, the most commonly known example being sunburn from UV light.
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Burns are classified by degree, where severity increases with a higher degree. A newer classification system separates burns into three categories: superficial, partial thickness and full thickness burns. The patient in the scenario has a full thickness third degree burn. This usually results in loss of the outer skin layer (epidermis) causing the patient’s skin to be numb and feel hard and leathery to the touch. This degree of burn requires immediate medical attention as they do not heal on their own. Proteins at the site of injury denature and cells eventually die, marked by the formation of black eschar at the centre of the wound. Once the skin barrier has been broken, the homeostatic functions of the skin are lost. There is rapid loss of body fluid and blood plasma which can sometimes cause visible swelling.
As the skin is the body’s first line of defence, burn wound infections correlate with the size of the burn injury. The epidermis constantly sheds off and takes harmful bacteria with it. Furthermore, the acidic nature of the skin prevents the entry of microorganisms that generally prefer neutral conditions. Burns are also associated with a depressed immune response. Given this information, it is no surprise that infection is the principal cause of death in burn victims. As it is a third degree burn, the patient will have spent some time in hospital where there is a greater risk of infection. Patient to patient contact via hospital equipment and staff allows rapid transmission of pathogenic bacteria. The burn wound is a favourable site for opportunistic colonization by pathogens because the eschar provides a moist, protein rich environment which encourages bacterial growth. The excess of necrotic tissue provides a rich growth medium for the microorganism.
In the clinical presentation, the patient complained of fever, chills and an unpleasant smelling discharge from the burn wound. These are all signs of infection so the doctor was correct to prescribe the patient an antibiotic. However, on the basis of the array of microbiology data collected in the laboratory, the doctor’s choice of antibiotic was incorrect. A two week course of flucloxacillin 500mg was prescribed to the patient to be taken orally 4 times daily. Flucloxacillin is an antibiotic belonging to the penicillin group. It is used to treat infections caused by gram-positive bacteria, most commonly Staphylococcus aureus. In the laboratory, the microscopic slide was examined under a microscope using the oil immersion lens. Rod shaped microorganisms were observed and they were stained a pink/red colour. This indicates the bacteria present in the pus samples is gram negative. Therefore, flucloxacillin would have no therapeutic effect to the patient as it is only bactericidal against gram-positive cells.
The three agar plates were examined and observations on the colour, shape, size and smell of the colonies was noted. There was no visible growth on the Mannitol salt agar (MSA) plate. This type of selective agar is used to isolate Staphylococcus aureus as it inhibits most other bacteria due to its high sodium chloride concentration (7.5 %w/v). The absence of growth on the MSA plate provides further evidence that there is no Staphylococcus aureus colonisation in the patient.
The colonies on the cetrimide agar were a pale, cloudy brown colour and gave off the smell of rotting fruit. The use of cetrimide agar as a selective and differential agar allows for the isolation of Pseudomonas aeruginosa, a gram-negative microorganism. Cetrimide is an ammonia compound which inhibits most bacteria, including some Pseudomonas species (but not P. aeruginosa). It also enhances production of the pigment pyocyanin which results in bright green colonies. However, some strains of P. aeruginosa do not produce the pigment. The growth on this plate confirmed the pathogen present in the patient is Pseudomonas aeruginosa and the colourless colonies indicate it is a non-pigment producing strain.
There was also growth on the MacConkey agar where the colonies were somewhat colourless but had an orange tinge. MacConkey agar is a selective differential medium used to identify gram-negative bacteria and determine whether they are lactose fermenters. Fermenters appear as bright red colonies whilst non-fermenters are relatively colourless. As there was growth on this plate, the microorganism is definitely gram-negative but not a fermenter due to the absence of red colonies. P. aeruginosa does not ferment lactose which explains the colour of the colonies.
An oxidase test was carried out which gave a positive result, indicated by a deep blue colour at the end of the test stick. An oxidase positive microbe possesses the enzyme required in the final stage of respiration to form metabolic water. Only aerobic or facultatively anaerobic microorganisms give a positive result. This biochemical test presents further evidence that P. aeruginosa is the infection causing bacteria because it is an obligate aerobe and therefore possess the cytochrome oxidase enzyme.
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Broth bottles containing growth medium, a bacterial culture and an antibiotic at five different concentrations (all in µg/ml) were observed. Cloudy broth dilutions indicated the bacteria was resistant and was able to grow. Clear broth dilutions showed the bacteria was sensitive to the antibiotic and its growth was inhibited. The bacteria was resistant to erythromycin at all concentrations used (1.25-20µg/ml). Erythromycin is a macrolide antibiotic related to penicillin. The results indicate it would have no therapeutic effect if prescribed to the patient. Amoxicillin showed antimicrobial activity against the microorganism at concentrations of 20µg/ml and 40µg/ml. However, at concentrations above 12µg/ml, the bacterium is considered resistant to amoxicillin so it has no therapeutic effect to the patient. Penicillin’s do not seem to have significant antimicrobial effects for this pathogen which shows the doctor was incorrect in his prescription of flucloxacillin as it is too a member of the penicillin group. The dose of 500mg will unlikely be high enough to be beneficial and any therapeutic doses would be at too high a concentration and therefore impractical.
Cefuroxime, a second generation cephalosporin, was effective at concentrations of 16µg/ml and 32µg/ml. Again, this was above the intermediate concentration range so does not benefit the patient. A newer generation cephalosporin may need to be used as they provide coverage against a broader spectrum of bacteria. The broth dilution containing ticarcillin was clear at concentrations of 8µg/ml and 16µg/ml. The intermediate concentration range for this drug is 10-20µg/ml so the minimum inhibitory concentration is 8µg/ml. This drug is a carboxypenicillin – a sub group of the penicillin family. The bacteria showed moderate sensitivity to the drug but only at fairly high concentrations. Amikacin was also effective below its intermediate range (6-10µg/ml) and inhibited bacterial growth at concentrations of 4µg/ml, 6µg/ml and 8µg/ml. Amikacin is an aminoglycoside and is a suitable antibiotic to use against this pathogen as it showed high sensitivity to the drug.
The doctor’s choice of antimicrobial therapy will not be beneficial to the patient as flucloxacillin is effective against gram-positive bacteria only and the patient is infected with gram-negative P. aeruginosa. Unnecessary use of this antibiotic could cause bacterial resistance. P. aeruginosa is naturally resistant to a wide range of antibiotics and thus infections where it is the causative agent can be difficult to treat. Broad-spectrum agents are required as they are the only drugs which provide coverage against the bacteria. The patient could be treated with an aminoglycoside such as amikacin. Gentamicin could also be used but the experimental data shows the pathogen is definitely sensitive to amikacin and studies have found that P. aeruginosa is more likely to develop resistance to gentamicin than amikacin. Aminoglycosides are bactericidal and act by interfering with the bacterial ribosome, leading to incorrect reading of messenger RNA. This would be used along with a third generation cephalosporin such as ceftazidime. Carboxypenicillins could also be used (e.g. ticarcillin) but they have only moderate activity against Pseudomonas species and research has shown that bacteria quickly develop resistance to them. Third generation cephalosporins provide coverage against P. aeruginosa, unlike 1st and 2nd generation which have a more limited spectrum of activity. They are also bactericidal and exert their effect by interfering with cell wall synthesis. Although there is no significant evidence of the benefits to using the two drugs in combination, it is rational to do so as it prevents the emergence of mutant genes resistant to one of the agents. Both drugs are also thought to have a synergistic effect when used together. Neither of these drugs are absorbed via the gastrointestinal tract so must be administered via the parenteral route. Amikacin is given via intramuscular injection at a dose of 15mg/kg daily in two divided doses. The dose must be decided carefully as high levels of amikacin in the blood stream can cause damage to the ear (ototoxicity). Ceftazidime is given by intravenous infusion at a dose of approximately 2g every 12 hours, depending on the severity of the infection.
In summary, the available microbiology data indicates that the doctor’s presumptive diagnosis was incorrect and that the patient has a burns infection caused by Pseudomonas aeruginosa. The absence of growth on the MSA plate disproved the idea of a staph aureus related infection. Gram-negative rods observed on the slide and growth on the MacConkey agar clarified that the microorganism was gram-negative. Growth on the cetrimide agar plate confirmed the presence of Pseudomonas aeruginosa in the patient’s pus sample. Examination of the broth dilutions helped determine a suitable combination of antibiotics for the patient to take. These would be need therapeutic and possess antimicrobial activity against the pathogen, unlike the doctor’s prescription of flucloxacillin. Use of a third generation cephalosporin with an aminoglycoside should effectively clear up the infection in the patient following completion of the course of antibiotics.
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