The development microbial colonies into biofilms on medical devices is a very serious problem for the medical industry, leading to infections that complicate medical problems and leading to longer hospital stays and more expensive treatments. In this investigation I used Klebsiella pneumonia, common cause of these infections, identifying and then treating with an antibiotic. Through this investigation I aim to identify the organism as Klebsiella pneumonia and then treat the organism with an antibiotic that will hinder it growth and thus the development of a biofilm. A variety of testing methods was used to identify that the sample was Klebsiella pneumonia, including a Biofilms method, a Gram staining method, an API test method, an MIC test method, an exposure to antibiotic Gentamicin (Susceptibility test) method and a Scan Electron Microscopy test method, and a sample of this was then treated with the antibiotic over a 14 day period. The results of the identification test showed the organism was able to form a biofilm, was Gram Negative, was sensitive to the antimicrobial agent used for the MIC test a could be identified through Scan Electron Microscopy. The API test confirmed that the enterobacteria we had was Klebsiella pneumonia. I found that the treatment was effective in controlling colony size, but was not able to achieve complete eradication. From this test I was able to conclude that the antibiotic was successful in controlling numbers after the development of colonies, but recommend that such treatments should be used cautiously as it could not be used to prevent infection entirely and could indirectly lead to the development of other infections. The development of strains that are resistant to microbes is an important concern, and suggests that such pathogens might be better controlled through strict hygiene measures and through the development of drugs that target pili rather than the whole of the cell and that the suitability of various artificial substrata for use in the manufacture of medical devices warrants further investigation.
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Keywords: Klebsiella pneumonia, Biofilms, HCAIs, Antibiotics, Treatment of Infection.
Artificial Substrata such as the materials from which medical implements are made can be very important in the growth and development of a microbial colonies and biofilms. Biofilms are defined as groups of microbes existing as communities on surfaces of medical devices1 and this makes them a significant source of HCAIs (Health Care Associated Infections) that cost health services billons every year, not to mention the discomfort that they give to a large proportion of Hospital patients (around 8% of those admitted in 2006 reported HCAI symptoms2). Biofilms are a serious problem because they are often resistant to antibiotics, causing the organisms growing on biofilms to be 1000 times more resistant to antibiotics than their counterparts not growing on biofilms3. The most common organisms to cause HCAIs are Staphylococcus aureus and Clostridium difficile, but Klebsiella pneumonia is also a significant source of infection and the biofilms associated with this organism will be the focus of this investigation. This organism, considered harmless in its natural surroundings and is normally found in water courses and sewage2, can cause serious infections such as bacterial pneumonia2 in the respiratory and urinary tracts. The resistance of biofilms to antimicrobial agents means they are an important cause of persistent infections4 and highlights the need for development of effective treatment against biofilms or to prevent their formation.
In this case K. pneumonia is being used a species from which general conclusions on treatment can be drawn, and it is hoped that success in this investigation can show us how many biofilms can be treated. Vast majority of Klebsiella spp. infections are associated with hospitalisation2 and K. pneumonia is considered to be the most important species in terms of the medical complications that it can cause, so this is a species of significant medical importance. There has been considerable research in this area, and it is known that the treatment of biofilms depends very much on the substrate on which they grow; this report will aim to be more definitive in its conclusions as regards the effect of substrate variation on biofilm growth .As an important cause of infection if we understood more about how to control the growth of biofilms we could also limit the prevalence of biofilms as a source of infection. K. pneumonia is also a species that has developed resistance to many ant-biotic strains, increasing the importance of research concerning this species.
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There are several facts that are known about the species, including that it gram negative organism2, so I expect that it will produce a negative result for this test. I also anticipate that the biofilms test will give a positive result.
Materials and Method
In order to detect bacterial growth with the highest degree of accuracy a number of techniques were used to identify the micro-organisms that formed colony:
This approach was used in order to assess the capacity that the substrate would have for the growth of biofilm colonies5. For each test substratum, copper or stainless steel, 20mm diameter section were added to each well of a six well plate, and 3ml of 1% w/v sterilised yeast extract broth plus the L-cysteine (YEB+) was added to each well in order to immerse the discs. A total of 100 µl of the test inoculum (1x105 cfu/ml) was added to each well and the plates left at room temperature for 1 hour. The discs were then ascetically transferred into new sterile six well plates, covered with 3ml PBS and agitated gently at 20rpm for 3 minutes to remove non-adherent cells. Each disc was then transferred to another sterile well and covered with 3ml YEB+. The plates were then sealed with paraffin and incubated at 36oC, with the media being replaced at 3 or 4-day intervals. The simplicity of this procedure is the principal reason for its enduring popularity5.
Gram staining method
Gram staining is a common technique in microbiological methods, identifying microbes as either gram positive or gram negative6. I started by taking a small sample from a broth culture and placing it on a glass slide, then drying and 'fixing' the slide. Each slide is then stained with Gentian Violet and subsequently treated with Gram's solution. Each slide is then washed with ethyl-alcohol after about 30 seconds and stained with fuchsin. This will stain gram-negative cells red, as opposed to the blue-black colouration of gram-positive cells6. But in order to see cells more clearly I then reapplied the fuchsin stain, then blotting off extra water carefully with absorbent paper.
API test method
API is a test used to identify mainly Enterobacteriaceae and is one of several systems available for this task7. The test requires 20 miniaturized compartments, each containing a dehydrated substrate for a different test. The API system is comprised of the following tests: o-nitrophenyl-fl-D-galactosidase (ONPG), arginine dihydrolase, lysine and ornithine decarboxylase, citrate utilization, H2S production, urease, tryptophan deaminase, indole production, acetoin production, gelatinase, and fermentation of glucose, mannitol, inositol, sorbitol, rhamnose, sucrose, melibiose, amygdaline, and arabinose6. Each compartment was inoculated a small amount of nutrient broth suspended in 4.5 ml of sterile, distilled water at pH 7.0 to a density of approximately 106 organisms per millilitre; McFarland barium sulphate standards were used for comparison. The compartments of the API system were then filled by using a Pasteur pipette. The strip containing the inoculated compartments was then incubated overnight at 37oC in the plastic container supplied by the manufacturer and with a small amount of water to maintain humidity. After 18 to 24 hr, results were recorded and identifications made and the API system was discarded. The results of this test and other tests can be used to confidently predict the micro-organism species.
MIC test method
The MIC (minimum inhibitory concentration) is the most commonly used value which indicates the sensitivity of an organism to an antimicrobial chemical. It is usually determined by inoculating a series of tubes of broth containing doubling concentrations of the chemical. At some point in the series the concentration is sufficient to inhibit bacterial growth, and this is referred to as the MIC. The apparatus that you will need for this test includes 30 sterile test tubes, iso-sentist broth, Gentamicin, a test-tube rack, a Gilson pipette, gloves, an agar plate containing TSA, an inoculation loop and a Bunsen burner. The first stage in the method is to label the first 2 test tubes as positive and negative, the rest are labelled from 8 to 1 as (64µg/ml), 32µg/ml, 16µg/ml, 8µg/ml, 4µg/ml, 2µg/ml, 1µg/ml and 0.5µg/ml) and then allocate K. pneumonia with the combination of gentamicin to each tube. After this pipette 5 ml of iso-sensitest broth without antibiotic into the positive and negative tubes only, as these two are your controls. Then add 5 ml of iso-sensitest broth with antibiotic to tube 8 using the Gilson pipette Take 2.5ml broth from test tube 8 and transfer it to test tube 7, and transfer 2.5m1 volumes down the series so that the concentration of antibiotic is halved each time. Discard 2.5ml liquid from the last tube so that the volumes are the same throughout. Add one drop of culture of the Klebsiella punemoniae to each of the tubes and gently mix them all again before placing for incubation overnight at 370C. This procedure should be repeated 3 times to ensure accuracy.
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Exposure to antibiotic Gentamicin (Susceptibility test) method
This is a method that is often used to identify clinically relevant bacteria8, in this case the growth of the organism in the presence of Gentamicin, a common antibiotic, although other substances can be used for a susceptibility test9. The first stage of this technique is to prepare Petri dishes containing multi-sensitest. The performance of the tests should be monitored by the use of appropriate control strains for this test the controls used were taken were Haemophilus influenzae NCTC 11931 and H. Influenzae NCTC 12699 (ATCC 49247)8. In preparation of the inoculums I selected only colonies that gave semi-confluent growth after inoculation, as these allow incorrect inoculums to be more easily observed. The plates were incubated at 37oC for 18h after inoculation and compared with a 0.5 McFarland standard.
Scan electron microscopy (SEM) method
To identify an organism that has produced a biofilm it may be feasible to examine an organism using SEM, the technique used here being one that preserves biofilms sufficiently to allow them to be observed using SEM. The adherent cells were fixed using 2.5% glutaraldehyde (manually prepared) in cacodylate buffer at 7oC for 1 hour to remove 'natural' water. The samples were rinsed three times in cacodylate buffer, and then dehydrated in increasing concentrations of ethanol (25%, 50%, 75% and two washes in 95%) Each wash lasted for 15 minutes, and was conducted at room temperature and atmospheric pressure. The final ethanol was poured off, and the sample freeze dried at -20oC. The samples were mounted, and sputter-coated with palladium, and then viewed by SEM using a JEOL JSM- 6310 electron microscope.
Once the organism identification procedure has been completed the next stage is to assess if the chosen antibiotic will be effective against the target organism, which in this investigation is K. pnuemoniae. Organisms were lyophilized and stored at - 15°C or frozen on agar and stored at -70°C until used10, and stored at 4oC during use. The samples were then incubated with the anti-biotic chloramphenicol for 48 hours at 37oC. The control for this experiment was a sample of K. pnuemoniae that was incubated without the anti-biotic. The significance of perceived differences between the two sample groups was analyzed with T-tests where appropriate. The formula that was used to calculate CFU/disc is
CFU/disc = overall average x 1/0.1 x 1x 10 to the power of the dilution
The methods detailed above indicated that the subject of this investigation was K. pneumonia and so subsequent treatments were administered on this basis. The results of the identification test showed the organism was able to form a biofilm, was Gram Negative, and was sensitive to the antimicrobial agent used for the MIC test. The API test confirmed that the enterobactria we had was Klebsiella pneumonia. Table 1.1 shows the results of the test resistance to Gentamicin and from this experiment it is found that in the liquid the microorganism is acceptable at 4 µg/ml, which the organism will grow when the concentration is less than this. The observations taken from SEM are shown in appendix one. Figure 1.1 shows no growth, only a rough surface can be observed, as is confirmed by the very low colony size seen on figure 2.5. When the control is observed after 14 days, it is seen from figure 1.2 that a large number of cells have formed an aggregate. Furthermore, the fact that there is evidence of cell division indicates that the cells are metabolically active. The hallowed effect of some cells could possibly be due to dehydration. The effect of the ant-biotic after 14 days can be clearly seen in figure 1.3. The colony has begun to recover from anti-biotic action as cell division is evidence of re-growth. The colony is smaller than in the control and there is evidence of the recovery suggested in figure 2.5.
The graph shown in figure 2.1 of the appendix indicates that in the first 5 days after incubation there is a significant decrease in the size of the bacterial colony. The graph shown in figure 2.1 shows standard deviations between the colonies under observation. The standard deviation that is shown indicates the variation between samples and therefore allows us to draw conclusions on the transferability of the treatments and the reliability of results. In this case the high SD after 3 days is to be expected due to variations in the rates of reduction, and the low amount of variation between samples shows that samples are experiencing similar levels of reduction and thus reinforces the reliability of results. Figure 2.3 demonstrates the growth of the non-resistant control, whereas figure 2.4 demonstrates that of the resistant control. Both show similar patterns of decrease to that of the target colony, suggesting that the bacteria of the target colony were not affected by the treatment.
The effect of the antibiotic treatment on colony growth is shown in table 2.1. From these tables we can see that growth of the colonies was unaffected during the first week, however antibiotic treatment results in significantly inhibited colony growth at the 11 (t-value = 7.87, p-value = 1.64x10-6) and 14 day (t-value = 5.748631797, p-value = 0.000128629) intervals. Tables 2.2 and 2.3 show p-values calculated at the 11 and 14 day intervals respectively and figure 2.5 shows graphically that there is a reduction in growth after approximately 7 days in response to the antibiotic. The MIC test indicated that K. punemoniae is resistant Gentamicin under the conditions of the test, and in addition to this an MBC (minimum bacterial concentration) was also conducted to determine the concentration of antibiotic that is lethal to the target bacteria in vitro. Through API 20 E tests I was able confirm that the organism being used for this investigation was K. pnuemoniae.
Misidentifications are common among Klebsiella spp.2 and so the establishment of a reliable identification method is important. The anti-biotic used against K. pnuemoniae in this investigation was effective in so far as it did inhibit the growth of the biofilm, but over the timescale of the experiment of the experiment it was not successful in eradicating the organism. Based on this data I would suggest that the treatment should be used to control a minor infection but I cannot say if it would ultimately lead to eradication; and due to the length of the post-inoculation ineffective period I do not think this would be a suitable anti-biotic for a serious infection. Nevertheless, K. pnuemoniae is an organism that is resistant to many anti-biotics, one of the reasons for its success as a HCAI, so it may to be useful to identify what could be a reasonably effective treatment, particularly as infections from this species are particularly prevalent among vulnerable groups, such as in paediatric wards2. The results from this investigation suggest that this antibiotic could be used where K. pnuemoniae is known or suspected to be the vector for infection. This method can also be used to confirm the presence of K. pnuemoniae on a substrate, allowing treatment prior to patient contact, thus preventing this possible avenue of infection.
This investigation may benefit from being conducted over a longer timescale as no effect was detected to the latter half of the experiment and this would allow more information to be collected on culture growth in the presence of the anti-biotic. This procedure could also be adapted to show how K. pnuemoniae might respond to other treatments. There could be a number of β-Lactam anti-biotics that could be effective against K. pnuemoniae and other organisms that can form biofilms, and if further treatments that could prevent HCAIs could be found this might be an important area for further research. There is growing resistance to β-Lactam anti-biotics in some strains of K. pnuemoniae2, so it could be useful to re-evaluate
the effectiveness of this group in treating cases of K. pnuemoniae.
It may also be possible to draw conclusions on the effectiveness of treatment from critical examination of the standard deviation of the readings. Figure 2 indicates that the greatest variation in recordings was observed after 3 days.
These bacteria have had success because of their resistant to anti-biotics; in fact many of the anti-biotics common in hospitals will aid the growth of K. pnuemoniae colonies2. As the use of anti-biotics can reduce their effectiveness, it is perhaps best to maintain good hygiene to prevent spread of the organism. It has been shown that anti-biotic treatments can be effective, but as their use must be limited to maintain effectiveness, strict hygiene may be the most effective treatment. In addition to hygiene it is important to ensure the appropriate use of artificial substrates, possibly avoiding those that have the greatest probability of harbouring harmful biofilms.
This investigation has shown that K. pnuemoniae can be successfully treated through the use of anti-biotics; however there are several factors that need to be considered to complete an evaluation of the treatment. Perhaps the most important consideration is that increased use of anti-biotics will ultimately lead to a decrease in their effectiveness as there will be an increase in resistance amongst the target population, and a possible increase in allergic reactions amongst patients. There is strong evidence to suggest that resistant strains are common in many micro-organism species11. Furthermore, the effect on the treatment takes several days and the results of this investigation do not suggest that complete eradication of the infection will occur. An administrator of the treatment should also consider if the patient has received any other antibiotic treatments recently as this could have a negative effect on the treatment11. Although the results have shown the treatment is effective, I feel that preventative measures are most important in this case.
There are several alternatives to anti-biotic treatment that can be used - such as adopting the strict hygiene regime that is mentioned above. It has been shown that many enterobacteria need to come into contact with the cell membrane prior to infection and complete such adhesion by the use of pili2, suggesting that future research might focus on measures to target the pili rather than inhibiting the growth of biofilms. There is also evidence to suggest that K. pnuemoniae may bind to sugar in the gut, suggesting that a low sugar diet may also be a suitable method to prevent infection. There are many methods that could be used as an alternative to anti-biotic treatment, the use of which should be limited in order to maintain effectiveness.
To prevent the longer hospital stays that are so often a result of K. pnuemoniae infection11, we need to quickly and effectively identify risk factors. Unnecessary use of anti-biotics needs to be reduced if we want to reduce HCAIs; infection is closely related to anti-biotic use11. If we reduce exposure through good hygiene this should have a significant effect, as multiple exposures are often needed for infection. Health care professionals may also wish to consider carefully the devices that they, ensuring that they are well informed of current advice on appropriate substrates, taking care to avoid those that may carry the biofilms of potentially harmful bacteria.