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Speed and cost are 2 vitally important factors in clinical microbiology. Patients increasingly expect test results returned in the shortest possible time, and hospital funding cuts dictate that low-cost tests are of major significance. Biochemical tests are therefore more commonly used than new molecular and genetic techniques in microbial taxonomy, as they have been found to be more time-consuming and expensive than standard biochemical tests.
The Gram's stain is the empirical method of differentiating bacterial species into two large groups (Gram-positive and Gram-negative) based on the chemical and physical properties of their cell walls. The Gram's stain is almost always the first step in the identification of a bacterial organism. From identifying whether the bacterial species is Gram-positive or Gram-negative, subsequent biochemical tests can be carried out to attain further identification.
One of the tests that can then be carried out is the Analytical Profile Index (API). This is a test that consists of a series of microtubules containing different dehydrated substances. A suspension of bacterial cells is then inoculated into the API plate, and is therefore rehydrated. Following a period of incubation, the substrate will react with the bacteria and if the bacteria is capable of metabolising the substrate, then a colour change will occur. There are different types of API plates that may be used for specific identifications. The API plate used in this investigation is the 20E API which is used for aerobic Gram-negative rods.
This is a table of the results found from the biochemical tests carried out on both bacterial cultures. Catalase and coagulase tests were only carried out on the Gram-positive bacteria, and the oxidase test was only carried out on the Gram-negative bacteria.
This is an image of the API plate innoculated, and the positive and negative results for each section.
This API result was fed into the computer system and the identification returned is Citrobacter freundii (99.8% ID)
1) Coagulase negative staphylcocci are considered as microorganissms with little virulence and are usually found as contaminants. The first bacteria isolated in this experiment fits into this catergory. Coagulase negative cocci are usally found in clusters, as I observed microscopically.
The major pathogen associated with coagulase negative cocci is Staphylcoccus epidermis, which has colonies which are typically small, white beige and about 1-2 nm in diameter. S. epidermis is also catalase positive, so fits with the results from this investigation. This bacterial species is considered to be non-pathogenic but patients, in hospitals for example, with comprimised immune systems, are often at risk of developing infection. Most infection from S. epidermis are nosocomial or community acquired. Hospitals often carry more virulent strains of the bacteria due to continuous use of disinfectants and antibiotics.
S. epidermis is a bacteria that grows biofilms on plastic and metal surfaces. This can cuase significant infection if these biofilms grow on medical devices such as hip joints or heart valves. These biofilms produce thick surfaces with many layers of the bacteria, which makes it hard for antibiotics to be effective when infection presents as they cannot effectively reach all bacteria.
S. epidermis, as a nosocomial pathogen, can cause sepsis in premature infantiles, urinary tract infections, eye infections, endocarditis, and infections associated with intravascular catheters.
The isolation of S. epidermis from clinical samples is of major significance. As S. epidermis effects primarily those with weakened immune systems in a hospital environment, this bacteria must quickly be isolated and idenntified so that the correct method of treatment can be given. If the bacteria is not identified, patients could suffer very significant infections without the capability in themselves to create an immune response.
Also, nosocomial infections are easily spread in the clinical environment. Once an isolation of S. epidermis is made in the laboratory, this suggests that there has been contamnation of hospital equipment, and it is then brought to the attention of clinical staff in that area. All equipment must be kept sterile to avoid further infection of patients.
Since antibiotics are largely ineffective in clearing biofilms, the most common treatment for these infections is to remove or replace the implant. In all cases prevention is ideal. Disease may be prevented by removing catheters and infected shunts. The drug of choice in treating S. epidermis infections is often vancomycin, to which rifampin or aminoglycoside can be added. If the bacteria is not isolated from sample, rounds of ineffiective treatment may initially be given to a patient, in which time the health of the pateint could deteriorate. Without an identification of the bacteruim, clinicians would not know to remove the implanted device which is causing the disease, and the problem would continue.
2) In this investigation, I used API 20E to identify my Gram-positive bacterium. These are the tests done, and the results they give:
Test Substrate Reaction Tested Results (positive) Results (negative)
ONPG ONPG Beta-galactosidase colorless yellow
ADH arginine arginine dihydrolase yellow red/orange
LDC lysine lysine decarboxylase yellow red/orange
ODC ortnithine ornithint decarboxylase yellow red/orange
CIT citrate citrate utilisation pale green/yellow blue-green/blue
H2S Na thiosulfate H2S production colorless/grey black deposit
URE urea urea hydrolysis yellow red/orange
TDA tryptophan deaminase yellow brown-red
IND tryptophan indole production yellow red (min 2 mins)
VP Na pyruvate acetoin production colorless pink/red (10 mins)
GEL charcoal gelatin gelatinase no diffusion of black black diffuse
GLU glucose fermentation/oxidation blue/blue-green yellow
MAN mannitol fermentation/oxidation blue/blue-green yellow
INO inositol fermentation/oxidation blue/blue-green yellow
SOR sorbitol fermentation/oxidation blue/blue-green yellow
RHA rhamnose fermentation/oxidation blue/blue-green yellow
SAC sucrose fermentation/oxidation blue/blue-green yellow
MEL melibose fermentation/oxidation blue/blue-green yellow
AMY amygdalin fermentation/oxidation blue/blue-green yellow
ARA arabinose fermentation/oxidation blue/blue-green yellow
OX oxidase oxidase colorless-yellow violet
In identifying Citrobacter freuindii the following tests were positive:
These positive results are important in the identification of this species, as it shows us what substrates can be fermented/oxidised, or reacted with. These ractions differ for all bacteria.
The oxidase test is a key test in the identification of this bacterium. Because it is negative, it means that the bacteria does not contain cytochrome c oxidase and therefore cannot utilise oxygen for energy production. Typically enteriobacteriaceae are OX-, which we have found.
The normal practice of detecting S. epidermidis is by using the Baird-Parker Agar with egg yolk supplement. Colonies appear small and black. They can be confirmed using the coagulase test. Increasingly, techniques such as real-time PCR and quantitative PCR are being employed for the rapid detection and identification of Staphylococcus strains. Normally sensitivity to desferrioxamine can also be used to distinguish it from most other staphylococci, except in the case of Staphylococcus hominis, which is also sensitive. In this case the production of acid from trehalose, by Staphylococcus hominis, can be used to tell the two species apart.
a) The oxidase test is used in microbiology to detemine if a bacterium produces certain cytochrome c oxidases. It uses disks impregnated with a reagent such as N tetramethyl-p-phenylenediamine (TMPD) or N,N-dimethyl-p-phenylenediamine (DMPD) which is also a redox indicator. The indicator is a dark blue to maroon color when oxidised and colorless when reduced.
Oxidases catalyse electron transport between substrates acting as electron donors in the bacterium and tetramethyl-p-phenylenediamine or dimethyl-p-phenylenediamine - a redox dye present as the hydrochloride or oxalate salt (the latter has a longer shelf-life). The dye is reduced to a deep violet-blue colour in the presence of oxidase enzymes.
Oxidase positive bacterium may utilise oxygen for energy production in the electron transport chain. Oxidase negative bacterium cannot utilise oxygen in this way.
b) Bacterial species may be differentiated on the basis of their ability to reduce nitrate to nitrite or nitrogenous gases. This is the biochemical pathway im which nitrate is reduced to nitrite, which can then further be reduced to nitric oxide, nitrous oxide, or nitrogen.
The nitrate reduction test is based on the detection of nitrite in the medium after incubation with an organism. If present in the medium, nitrite will react with sulfanilic acid (Nitrate reagent A) to form a colorless complex (nitrite-sulfanilic acid). This complex will then yield a red precipitate (prontosil) when nitrate reagent B (alpha-naphthylamine) is then added to the test, as shown below.
A red color will be produced in the medium only when nitrite is present in the medium. Lack of a red color in the medium after the addition of sulfanilic acid and alpha-naphthylamine means only that nitrite is not present in the medium.
c) The ONPG test uses the substrate 0-nitrophenyl-ß-galactopyranoside to determine the presence or obsence of the enzyme ß-galactosidase in an organism. The test is important in differentiating among the Enterobacteriaceae which are commonly classified according to their ability to ferment lactose. It is also used to differentiate Neisseria lactamica from other fastidious Neisseria species.
The presence of two enzymes, permease and ß-galactosidase, are required to demonstrate lactose fermentation. True lactose non-fermenters do not possess either of these enzymes. Late lactose fermenting organisms do not have permease, but do possess ß-galactosidase which hydrolyses lactose to form galactose and glucose. ONPG is similar in structure to lactose. If ß-galactosidase is present, the colourless ONPG is split in to galactose and o-nitrophenol, a yellow compound.
If an organism posses B-Galactosidase, the enzyme will splits the B-Galactosidase bond, releasing of o-nitrophenol yellow color compound. If organism lacks the enzyme, the Galactoside bond remains intact and the medium remains colorless.
Positive: yellow color within 4 hours
Negative: colorless at 4 hours
d) The indole test is a biochemical test performed on bacterial species to determine the ability of the organism to split indole from the amino acid tryptophan. This division is performed by a chain of a number of different intracellular enzymes, a system generally referred to as "tryptophanase."
Indole is generated by reductive deamination from tryptophan via the intermediate molecule indolepyruvic acid. Tryptophanase catalyzes the deamination reaction, during which the amine (-NH2) group of the tryptophan molecule is removed. Final products of the reaction are indole, pyruvic acid, ammonia (NH3) and energy. Pyridoxal phosphate is required as a coenzyme.
Pure bacterial culture must be grown in sterile tryptophan or peptone broth for 24-48 hours before performing the test. Following incubation, add 5 drops of James'/Kovac's reagent (isoamyl alcohol, p-Dimethylaminobenzaldehyde, concentrated hydrochloric acid) to the culture broth.
A positive result is shown by the presence of a red or red-violet color in the surface alcohol layer of the broth. A negative result appears yellow. A variable result can also occur, showing an orange color as a result. This is due to the presence of skatole, also known as methyl indole or methylated indole, another possible product of tryptophan degradation.
5) 5). PCR could be used to test for microbes, this is where the bacteria will be broken up into genetic pieces and using a number of techniques to expand the dna by annealing and reheating to break it will allow for better testing strands of dna that can be probed to find specific 'flagship' dna sequences for the bacteria.
Agglutination test stimulates the antigens of a bacteria, it is placed in saline and mixed with antisera that has been cultured agaisnt a known bacteria, the test is positive if the bacteria clump together; Used to distinguish between different strains of the same bacteria.
Phage typing is another technique in which the bacteria is identified according to their response a viral phage, phages are specific to bacteria, the phage will be placed with thebacteria growing on a plate, where the bacteria don't grow you know it is not immune to that specific phage and therefore that bacteria.
Electrophoresis could also be used to analyse the protein stimilarites between organisms, the pattern branded at the end will be very similar for most bacteria.
Finally a urease test could be used to see secrete if the urease enzyme, which catalyzes the conversion of urea to ammonia and bicarbonate will affect growth
1). Based on the experiments carried out, I would reccomend treatment for infection caused by ps. aeruginosa with antibiotic ciprofloxacin. Cefuroxine has no effect on the bacteria, as there is no zone cleared at all. Contrastingly, ciprofloxacin has the greatest diameter of bacteria cleared, and is the most sensitive to the antibiotic. Piperacillin would also be a suitable antiobiotic for treatment of this bacteria, but not as effective as ciprofloxacin. Administeration of this antiobiotic would be effective on a higher proportion of bacteria in the body than any of the other antiobiotics tested.
2). The mic for impipenem on E.coli was 0.19, which meant it was senstitive to the antibiotic used.
3a). The results of this investigation show me that the depth of agar has an effect on the size of agar diffusion by antiobiotics. In nearly all cases, the size of the zone surrounding each antibiotic disc decreases as the depth of agar incraeses. Agar's structure is make up of a linear polymer made up of a mixture of agarose and agaropectin. Its' structure dictates that antiobiotics will first diffuse downwards, and then across the agar plate. An agar depth of 35ml means that the zone of diffusion is lower than that of the 25ml. This means that a lot of the antibiotic has diffused downwards, and the result we are given is not a clear indication of whether the bacteria is resistant or sensitive to the antibiotic. A swallow agar depth means that the antibiotic does not diffuse downwards at all. This leaves the plate very difficult to read, as zones merge into one another, and it is difficult to interpret where zones end. BSAC reccomended depth agar is a constant, and is effective as all measurements will be constant when the same depth of agar is used. All zone measurements will not be affected by depth.
3b). The dilution has affected the radius of the antibiotic resistance which greatly affects the treatment of a patient and that is why the 1.0x10(5) cfu/ml is used, the weaker solution would make more strains seem that they are more sensitive to an antibiotic than they are which in contrast using a heavier dilution would cause the effectiveness of the antibiotic to seem less observable. In nearly all cases the zone area increases as the dilution is less concentrated. This is due to the fact that there is less bacteria in a set area, so antibiotics can spread over a larger area. Changes in concentration of solution makes the difference between an antibiotic being classified as resistant or sensitive.
3c). Depth of agar and dilutions of specimen are not controlled, this could have consequences on the method of treatment for the patient. Variation in these 2 factors will give greatly varying results for the clearing zone diameters. This could mean a patient could be traeted with a certain antibiotic because there was a large clear zone around the antibiotic disk. It could be considered that a bacteria is sensitive to a certain antibiotic, due to measurement of zone diameter. However, this large zone depth could be attributed to a shallow agar depth, or weak concentration of sample. This could result in a patient being treated with the wrong antibiotic and having no effective result.
3) There was variation in the results of these 2 experiments, even though there was the same agar depth and the same innoculum concentration. This could be attributed to:
Error in innoculating the plates by hand - may not get complete coverage of the plate - semi confluent growth.
The dilution factor was done between a range of figures, so the figure we picked might not be accuartely repeated.
Bacteria may not be dispersed evenly throughout the sample, so there could be a higher concentration of bacterial cells in one plate to another.
The placing of the antibiotic disks was done by hand so the zones could overlap more on one plate than on another, due to the disks being closer to each other.
The method of measuring the diameter of the zones was inaccurate, as it was done by taking a ruler to the zones and measuring by eye. This will naturally cause great variation in measurements.
4). Blood agar plates are often used to test for microorganisms of a pathogenic nature. Blood agar normally hasa concentration for 10-15% mammalian blood, often using sheep or horse blood but for some cases require the use of human blood. Blood agar tests for fastidious organisms that require. Blood agar is useful in detecting where colonies have grown as often the result of the pathogen is the cause of haemoglobin lysis using Î²-hemolytic enzyme.
5).Synergistic activity is the activity in which two antibiotics will be taken and the effect of one will greatly enhance the effectiveness of the second antibiotic. Additive activity is when two or more chemicals equal the sum of their individual original effects. Anatagonistic is when two or more chemical substances on a biological system, the combined effect is less than it should be due to them working against one another.
Antagonism occurs between penicillin and erythromycin.