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Determining the Identity of two Unknown Bacteria Using Tests to Explore Fermentative abilities, activity of enzymes, and biochemical reactions Section: 002 11/13/2009 Ryan Heasley

Abstract The focus of this experiment was to identify two unknown bacteria, 17 A and 17 B. The identification of unknown bacteria produces benefits for many aspects of the research of microorganisms and helps physicians correctly treat patients. Multiple tests were performed to provide the fermentation abilities, presence of certain enzymes, and certain biochemical reactions. Qualitative observations were made on the tests, which were compared to an unknown bacteria identification key to aid with the identification process. The data provided sufficient results to suggest that the identity of Unknown 17 A was the gram positive bacterium, Corynebacterium pseudodiphtheriticum and unknown 17 B was determined to be the gram negative bacterium, Enterobacter aerogenes. Both bacteria are pathogenic and are commonly identified as the cause of infections in the human body. Although the tests used during the experiment were somewhat outdated and tedious, the tests still provided an inexpensive yet effective method to identify unknown bacteria.

Introduction There are many practical applications for identifying unknown bacteria. During research, it is of utmost importance to be able to identify unknown bacteria if diversity is being studied. Trying to identify unknown bacteria can also lead to the discovery of new species, for example, if tests are performed to determine the identity and the bacterium does not match any previous single species tests, it is possible that a new species has been discovered. Another very

important application for identifying unknown bacteria is found in medical laboratories. One of the main responsibilities of a medical lab is to determine the identity of pathogenic bacteria. In many cases it is very important to determine the specific type of bacterium causing disease so the physician is able to correctly treat the patients. The structure of bacteria plays an important role of what antibiotics work and which do not. The chemical reactions of the bacteria also play a huge role in the usefulness of antibiotics. Most antibiotics alter or inhibit protein structure, inhibit transcription, inhibit translation, affect cell membrane structure, or alter cell-wall synthesis (1). Bacterial resistance is another pertinent medical reason for identification. The evolution of bacteria has made treatment of diseases much more difficult, which has been amplified by the improper use of antibiotics (1). Therefore, it is important to

know the chemistry, structure, and resistance of the pathogenic bacteria. A burn victim was being treated at a hospital and suddenly contracted a life-threatening infection. The symptoms of the patient included seepage of puss from the wounds and trouble breathing from a lung infection. The symptoms alone were not enough to identify the bacteria and due to the severity of the health of the patient, it was important to determine the exact pathogenic bacteria to limit the amount of unnecessary antibiotic use, which could be harmful to the patient. Unfortunately, there were no more Analytic Profile Index (API) test strips left in the

hospital, which have been used to identify unknown bacteria since 1986 (2). It was not legal to ship tests between hospitals and new shipments from the supplier were not expected for at least a week, so another plan of action needed to be implemented as soon as possible to help save the patient's life. The medical laboratory contacted a local college and was able to obtain tests to help aid with the identification. The tests studied the ability of fermentation for multiple sugars, the presence of different enzymes, and other biochemical reactions. Materials and Methods Two unknown bacteria were isolated from infections of the patient. Aseptic technique was practiced throughout the testing process. Constant bacterial cultures were maintained

throughout the experimentation and incubations for all tests were conducted for 48 hrs at 37°C, unless otherwise specified. An identification key with all of the performed tests was used to determine the identity of the unknown bacteria (3). A Gram Stain was implemented to determine whether the bacteria were positive or negative. A drop of distilled water was added to a microscope slide and the bacteria were applied into the water and spread thinly along the entire area of the slide. Once the water dried, the slide was heat fixed by quickly moving the slide through a Bunsen burner three times. Crystal violet was applied to the slide for one minute, and then rinsed with distilled water. Grams Iodine was applied to the slide for one minute and rinsed with distilled water. The slide was rinsed with 95% ethanol for five seconds and immediately rinsed with distilled water. Safranin stain was added to the slide for two minutes followed by a rinsing with distilled water. A light microscope was used at 400x magnification to observe the stained slides. A negative stain was practiced to provide the cellular shape of the bacteria. The bacteria were spread on one end of a microscope slide. One drop of Negrosin was added on top of the

bacteria. Another slide was placed into the Negrosin at a 45° angle and was pulled across the length of the slide until the entire bottom slide was covered with stain. The Negrosin was allowed to dry and the shape of the bacteria was observed under 400x magnification. The bacteria were inoculated into glucose, sucrose, and maltose broth tubes by gently shaking a loop with bacteria in the top of the broth. Each tube was prepared with a Durham tube, phenol red, and either glucose, sucrose, or maltose. A positive result of the test was indicated by the color change from red to yellow, suggesting that an acid product was produced during fermentation. Another indication of a positive result was provided if the Durham tube entrapped gaseous CO2 product of fermentation. A weaker fermenter was demonstrated by a color change to orange. A triple sugar iron agar (Kligler) test was prepared as a agar slant and contained 0.1% glucose, 1.0% lactose, 1.0% sucrose, ferrous ammonium sulfate, and phenol red. The bacteria were streaked on the surface of the agar with a loop and stabbed down through the middle of the agar to the bottom of the slant with a needle. Indication of glucose fermentation was displayed by the color change of the butt of the agar from red to yellow, due to acid production, and/or the formation of a bubble below the agar from CO2 production. A positive result for lactose or sucrose fermentation was indicated when the entire tube turned yellow from the presence of acid and/or cracks formed throughout the agar from the release of CO2. Weaker fermenters were displayed by an orange color change. The breakdown of ferrous ammonium sulfate was

indicated when a black precipitate, H2S, formed in the agar. A starch plate was used to test for amylase activity. Both bacteria were applied to the starch plate by streaking one single line for each bacterium about an inch apart on the gel. The

starch plate was flooded with Grams iodine and decanted after a few seconds. A positive test result for amylase activity was displayed by a fairly large clearing around the bacterium, which was created from the hydrolysis of starch by amylase. Urease activity was tested by inoculating the bacteria into tubes prepared with phenol red, urea, and a buffering system. The tubes were incubated overnight and observed within 24 hrs. A positive indication for urease activity was the color change from orange to hot pink due to the production of an acidic product. To test for the presence of gelatinase, a tube with gelatin, peptone, and beef broth was stab inoculated. Before the test was observed, the tube was placed in a refrigerator for 30 mins at 9°C. If the solid gel turned to liquid, the breakdown of gelatin from gelatinase activity was demonstrated. A Dnase test was utilized to assay for the Dnase activity. A nutritive agar plate

containing peptides, methyl green, and an emulsion of oligonucleotides was streaked in a single line from one end of the plate to the other with both types of bacteria about one inch apart on the plate. A clearing around the bacteria indicated that DNA was broken down by the activity of Dnase. To test for flagullar bacterial movement, a motility test was implemented. The test used was comprised of 0.5% semi-solid agar. An inoculation needle was used to pick up a small amount of bacteria and stab directly in and out of the middle of the semi-solid agar. If the bacteria moved outward from the stabbed area, a positive for motility was recorded. The indole test was performed to study the ability of the bacteria to break down tryptophan with tryptophanase. The liquid media contained 1% tryptone from the pancreatic

digest of the milk protein casein. Five hundred µL of the Kovacs reagent were added to the tubes and shaken very gently. A red ring at the very top of the broth indicated a positive result for the presence of indole and tryptophanase activity. To prepare the methyl red and Voges-Proskauer tests, the bacteria were inoculated into a media of mixed acids and butanediol. The tubes were incubated for 48 hrs at 37ºC. Three µL of the culture was transferred to a sterile tube to add two Barrit's reagents for the Voges-Prokauer test, which looks for the fermentation of butanediol. Six hundred µL of alpha-napthol was added followed be the addition of 200 µL of potassium hydroxide. A positive result was represented by a color change to red, indicating the presence of acetyl methyl carbinol. In the original test tube, 200 µL of methyl red was added. The positive result was indicated by a color change to red, which shows lactate, succinate, and or acetate were produced from the mixed acid fermentation. The presence of citrate transporters was studied using the Simmons citrate test, which used a synthetic medium composed of mineral salts, ammonium ions, and citrate. Brom thymol blue was the pH indicator used in the test and a positive result for citrate transporters was shown from a color change from green to blue. The test was inoculated in the same manner as the Kligler test. A catalase test was implemented to demonstrate catalase activity. A loop was used to pick up and spread a small amount of bacteria onto a microscope slide. Two drops of hydrogen peroxide was added on top of the bacteria. The production of bubbles indicated that catalase activity was occurring. To observe if the bacteria had cytochrome C, an oxidase test was employed. A toothpick was used pick up bacteria and scrape onto filter paper. Three drops of

tetramethylphenylenediamine was added to the bacteria. A positive result for cytochrome C was indicated if the tetramethylphenylenediamine turned from clear to purple when

tetramethylphenylenediamine donated electrons to cytochrome C. Results The morphological characteristics of unknown bacterium 17 A consisted of a white color, entire margin, and convex elevation. The Gram Stain suggested that the unknown 17 A was gram positive. The negative stain proposed the shape of the individual bacterium to be rod-like. The glucose, sucrose, maltose, Kligler, Dnase, citrate, and catalase test all provided positive results. The identity of the bacterium 17 A was determined to be Corynebacterium

pseudodiphtheriticum (Table 1). Unknown bacterium 17 B displayed a white-clear color, irregular margin, and convex elevation. The bacterium was determined to be gram negative, which was indicated by the Gram Stain. The negative stain suggested the shape of the bacteria to be rod-like. The tests of motility, glucose, sucrose, maltose, Kligler, citrate, and catalase all produced positive results. The most plausible identity of bacterium 17 B was determined to be Enterobacter aerogenes (Table 1). Any test not mentioned produced negative results. Discussion The Identity of unknown bacterium 17 A, Corynebacterium pseudodiphtheriticum, was supported by all of the tests performed during the experimentation (3). Some of the tests provided more implications than others. The search was immediately narrowed by the fact that the bacterium was rod-shaped and gram positive. The phenol red sugar tests help narrow the search even further to just Corynebacterium pseudodiphtheriticum and Lactobacillus

acidophilus. The Kligler test pointed toward C. pseudodiphtheriticum because L. acidophilus is

a more vigorous fermenter of glucose and sucrose and would have turned the agar completely yellow. The catalase test was also vital because C. psuedodiphtheriticum displays catalase activity, while L. acidophilus does not (Table 1). The Identity of unknown bacterium 17 B was much more difficult to designate. Not all of the tests were congruent with the identification key of bacteria (3). The Voges-Proskauer and Dnase tests did not coordinate with the expected results for Enterobacter aerogenes, but all of the other tests matched, which led to Enterobacter aerogenes as the identity of unknown 17 B. The Kligler test provided beneficial data that limited the search down to E. aerogenes, Escherichia coli, and Klebsiella pneumoniae because strong fermentation occurred, but the breakdown of ferrous ammonium sulfate did not arise. E. coli was eliminated from the

discussion because the citrate and methyl red tests did not match the experimental results. K. pneumoniae and E. aerogenes only differed in the motility test. The motility test was performed two times to ensure the legitimacy, which provided that the motile bacterium E. aerogenes was identified (Table 1). Corynebacterium pseudodiphtheriticum is closely related to many more familiar bacteria, such as diptheriae, Actinomyces, and actinomycosis, which are more commonly considered pathogens (4). C. pseudodiphtheriticum is often overlooked as a pathogen but does cause infections (5). The locations of infection are most common in the urinary and respiratory tracts and at intravenous sites. The bacteria tend to infect people who already have a pre-existing medical condition, such as renal disease, AIDS, tuberculosis, cancer, or anyone with a weakened immune system. Most strains of the bacterium are resistant to oxacillin, erythromycin and clindamycin. (6). The infections caused from C. pseudodiphtheriticum can be treated with many

antibiotics including penicillin, amoxicillin, cefamandole, cephalexin, rifampicin vancomycin, ciprofloxacin, and Tobramycin (7). Enterobacter aerogenes live in almost any environment including soils, water, the gastrointestinal tract of humans and other mammals, sewage, and in association with plant material and foods. E. aerogenes causes many different types of infections in humans.

Pneumonia, urinary tract infections, wound and burn infections, infections of intravascular and other prosthetic devices, and meningitis are all commonly caused by E. aerogenes. Hospitals tend to be a very popular place for these infections to arise and transmission of E. aerogenes is very ordinary between patients. E. aerogenes is completely resistant to ampicillin and first- and second-generation cephalosporins as a result -lactamase. Common mutations in the genome produce high levels of -lactamase, conferring resistance to third-generation cephalosporins (8). At this time, it is unclear what the optimal antibiotic treatment is for infections caused by E. aerogenes. In severe infections, physicians usually treat patients with combination antibiotic therapy to ensure the entire infection is eliminated (9). The tests used throughout the experiment may be a bit outdated, but they can and still do have applications in the world today. Microbiology students and laboratories use the tests and can be used in medical labs if needed. A better method for identifying unknown bacteria is to use API testing strips. The API test is a much more efficient because it is much faster and all tests are performed on a single strip that can fit in the hand. The use of the API tests is also of huge importance in the medical field (2). The Proper identification of infectious bacteria helps doctors determine the correct antibiotic treatment, which saves many lives.

Table 1. Tests and observations to determine the identity of the unknown bacteria Test/Observation Color Elevation Margin Shape Gram stain Motility Glucose brothA Maltose brothA Sucrose brothA KliglerB Gelatinase Starch hydrolysis Dnase Urease Indole Methyl red Voges-Proskauer Citrate Catalase Oxidase Identity

A

Unknown Bacterium 17 A White Convex Entire Rod + 1 1 1 1 + + + Corynebacterium pseudodiphtheriticum

Unknown Bacterium 17 B White Convex Irregular Rod + 2 2 2 2 + + Enterobacter aerogenes

1 = weak fermentation and color change to orange, 2 = strong fermentation and color change to yellow with gas in Durham tube B 1 = weak fermentation of glucose and a color change to orange, 2 = complete fermentation with a color change to yellow and cracks in the agar

Works Cited (1) Madigan, M. T., Martinko, J. M., Dunlap, P. V., & Clark, D. P. (2009). Biology of Microorgansims (twelrth ed. , pp. 791-803). San Francisco: Pearson Benjamin Cummings. (2) Our History (2009). Retrieved November 10, 2009, from http://www.biomerieux.com/servlet/srt/bio/portail/dynPage?node=Our_History (3) Potter, Beth (2009). Unknown Identification key. (4) Bacteriological and clinical aspects of corynebacterium (1989, June). Retrieved November 9, 2009, from PubMed. (5) Colt, H. G., Morris, J. F., Marston, B. J., & Sewell, D. L. (1991, February). Necotizing Trachetis Caused by Corynebacterium Pseudodiphtheriticum: Unique Case and Review. Retrieved November 9, 2009, from JSTOR. (6) Camello, Souza, Martins, Damasco, Marques, Pimenta, et al. (2009, February 18). Corynebacterium pseudodiphtheriticum isolated from relevant clinical sites of infection: a human pathogen overlooked in emerging countries. Retrieved November 12, 2009, from PubMed. (7) Endocarditis due to Corynebacterium pseudodiphtheriticum: five case reports, review, and antibiotic susceptibilities of nine strains (1992, December 15). Retrieved November 9, 2009, from PubMed. (8) Mandell, Douglas, & Bennett (2009). ENTEROBACTER SPECIES AND PANTOEA (ENTEROBACTER) AGGLOMERANS. Retrieved November 9, 2009, from MD CONSULT. (9) Marcon, M. J., & Cunningham, D. J. (2008). MICROBIOLOGY AND EPIDEMIOLOGY. Retrieved November 12, 2009, from MD CONSULT.

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