Isolation and identification of two unknown bacteria ABSTRACT Two different bacterial species were isolated and characterized by both performing microscope observations (to assess the morphology) and by using biochemical tests which could help identify the physiological and metabolic characteristics of the unknowns. By doing this it was possible to narrow down the identity of both samples A & B to B. laterosporus and S. choleraesuis, respectively. INTRODUCTIONThe goal of this experiment is to isolate and identify two different bacteria based on their morphological, physiological and metabolic characteristics, to this end, several biochemical tests will be performed to correctly identify the unknown bacteria down to the genus and species level. The identification and characterization of bacteria is a vital component in microbiology in both the scientific research and clinical settings because they provide valuable information regarding the microbial ecology of a specific environment, the metabolic capabilities of a bacteria, their pathogenicity or lack of, etc. This is more evident in the clinical field, in which the quick identification of bacteria allows physicians to save lives by: 1) providing them with the information required to make a more accurate diagnosis; 2) prescribing an appropriate treatment; 3) assessing the effectiveness of a treatment. Nowadays, the identification of unknown bacteria by using biochemical and/or physiological tests is considered for the most part obsolete because it is a process that can be both labor intensive and time consuming (days to weeks). Additionally, the test results are never guaranteed to be completely accurate since bacteria mutate constantly to adapt to their environments, thus they can occasionally express genes that change a key biochemical or morphological characteristic, rendering this technique inadequate (Rosselló, Amann 2001). Even so, in the clinical setting, in which speed is paramount, biochemical tests (coupled with other immunological techniques) are often the preferred way to quickly assess whether a known specific bacterium is present or absent in a sample. Currently, the most effective way of identifying the taxonomy and phylogeny of an unknown bacterium relies on the use of 16S rRNA gene sequences, which is a common genetic marker present in almost all bacteria (Janda, Abbott 2007). A disadvantage of this methodology is that it is costly and can be slow, since it usually involves sending the samples to a third-party biotechnology company that will perform the sequencing and analysis (Janda, Abbott 2007). Thus, in the following experiment we will first attempt to isolate two different microbial species, then proceed to characterize them by performing biochemical and physiological tests that will allow us to narrow down the identity of the bacteria. METHODS/MATERIALS Initially both unknown bacterial cultures were streaked on different TSA plates and then incubated at 37°C for 24 hours. Both plates are observed the next day to look out for visually different isolated colonies (since they are mixed bacterial cultures), these are then streaked using a loop onto a new set of Trypticase-Soy agar (TSA) plates and incubated as last time. After the initial setup is done, if the isolation was successful each TSA plate should have a single bacterium species (no mixed cultures), which can then be characterized using the Gram stain procedure. This straining technique can distinguish specific cell wall properties (i.e. presence or absence of LPS), which can help to narrow down the identity of the unknown bacteria. Bacteria that stain purple are Gram positive, and pink-looking bacteria are Gram negative, this not only categorizes the bacterium but also confirms whether the culture is pure or mixed based on the observed colors. Obtaining a single, isolated organism from the TSA plate is necessary to avoid variable results. After confirming with the Gram stain procedure that both cultures are pure isolates, both are then streaked onto a Trypticase-Soy agar slant (TSA slant) to allow for its growth during incubation at 37°C for one to two days. Once both cultures have been confirmed as pure and then identified as either Gram positive or negative, we can proceed to follow the bacterial identification dichotomous keys ( Figures 1 & 2) and perform the required biochemical tests to successfully narrow down the genus and species of both bacteria. The tests that were done to isolate both bacteria can be followed in Figures 1 and 2 by the black highlighting. The equipment and biochemical tests that were needed to finish the experiment are condensed in Table 1. For the sake of clarity, the order in which the tests were done and their results, along with the explanation as to why they were performed will be under the Results section, the next paragraphs will focus on providing information regarding the biochemical tests themselves, as well as how to interpret their results. 1) All sugar tests (glucose, lactose, sucrose, etc.): Tests for the production of gas when the sugar is fermented. A positive test will turn the media yellow because the fermentation lowers the pH, a negative test will stay purple. The presence of gas can be confirmed if bubbles are present within the Durham tube (bubbles should account for at least 10% of the volume of this inner tube to be considered positive). 2) Citrate test: Tests for the ability of a bacteria to transport and catabolize citrate as a carbon source under aerobic conditions. If the slant turns blue this accounts for a positive result (the bacteria can use citrate), however if it stays green it will be negative. 3) Nitrate reduction test: Determines if a bacterium can reduce nitrate (NO 3) to nitrite (NO2) using the enzyme nitrate reductase. It also tests if bacteria can perform nitrification on nitrate and nitrite to produce molecular nitrogen. If the liquid turns red this stands for a positive reaction (bacterium has the enzyme), while if it remains of the same color it accounts for a presumptive negative result. To further confirm if the result is positive or negative, zinc powder can be added to the liquid culture, if it turns red then it will be a confirmed negative, while if it doesn’t change its color it means that it is positive for the presence of the enzyme. 4) Bile Esculin agar (BEA): Used to confirm the presence of Enterococcus and certain strains of Streptococcus based on their ability to hydrolyze esculin. If the bacterium can hydrolyze esculin, the media will turn dark brown or black. The test is only considered positive if more than half of the slant is dark brown or black after incubation. 5) Methyl-red (MR) test: Indicates that a bacterium can ferment glucose by the accumulation of acidic products like acetate, succinate and formate, which will acidify the media, turning it red (positive reaction). No change in the coloring is considered a negative reaction. 6) Urease test: Used to test if the bacterium can degrade urea by utilizing the enzyme urease to hydrolyze the compound into ammonia and carbon dioxide. A positive reaction would require the accumulation of ammonia which will change the media to red, a negative reaction would stay yellow. 7) Kliger’s Iron Assay (KIA): The four biochemical properties that can be evaluated with a KIA test are: H2S production, lactose and/or glucose fermentation and the production of gas. Of the four, we will only assess whether the bacterium can produce H 2S, a positive reaction will be distinguished by the formation of a black precipitate in the lower portion of the slant. 8) Catalase test: Tests for the presence of the catalase enzyme in the bacterium. One drop of hydrogen peroxide is added to a wet-mount of the unknown, if any bubbling is observed it is considered a positive reaction. 9) Oxidase test strip: Tests if an organism is aerobic, the presence of a violet color in the paper stands for a positive reaction. Table 1: Biochemical tests and equipment used for the experiment Catalase test + - B. cereus, A. viridans, L. plantarum, L. lactis, S. salivarus, S. agalactiae B. subtilis, B. laterosporus, E. faecalis, S. aureus, S. epidermidis, S. saprophyticus Oxidase test MR test + - B. subtilis, B. laterosporus Sucrose test -/A/- B. cereus, L. lactis, S. salivarus, S. agalactiae MR test A. viridans, L. plantarum BEA test - + B. subtilis - + E. faecalis, S. aureus, S. epidermidis, S. saprophyticus - + B. laterosporus E. faecalis, S. epidermidis, S. A viridans Nitrate test - + S aureus E - + S. saprophyticus S. BEA test - + B. cereus, L. lactis S. salivarus, S. agalactiae Nitrate test ADC test + B cereus + - L. lactis S. salivarus S. agalactiae Figure 1: Dichotomous key for Gram (+) bacteria Catalase test + - C. freundii E. aerogenes, E. cloacae, E. coli, P. mirabilis, P. vulgaris, S. choleraesuis, S. flexneri, S. sonnei, S. marescens, K. pneumoniae, M. morganii Oxidase test - + E. aerogenes, E. coli, P. mirabilis, P. vulgaris, S. choleraesuis, S. flexneri, S. sonnei, S. marescens, K. pneumoniae, M. morganii E. cloacae Lactose test A/G -/- E. aerogenes, E. coli, K. pneumoniae P. mirabilis, P. vulgaris, S. choleraesuis, S. flexneri, S. sonnei, S. marescens, M. morganii Sucrose test -/- A/G E. aerogenes, K. pneumoniae P. vulgaris A/- li A/- P. mirabilis, S. choleraesuis, S. flexneri, S. sonnei, M. morganii Urease test + Sucrose test -/- A/G S. marescens E. aerogenes K. pneumoniae Urease test + S. choleraesuis, S. flexneri, S. sonnei, M. morganii - Citrate test P. mirabilis + S. choleraesuis S. flexneri, S. sonnei, M. morganii Nitrate test - + S. flexneri, M. morganii S. sonnei Glucose test A/G A/S. flexneri M. morganii Figure 2: Dichotomous key for Gram (-) bacteria Sample A was isolated from a mixed culture and then streaked RESULTS onto a TSA plate. The colonies of the A sample are round & smooth and present a white cream color, which can be observed in Figure 3B. Figure 3: A) Gram stain of unknown; B) Colony morphology of sample A. B Afterwards, by both visualizing the sample under the microscope and by performing a Gram stain, it was possible to conclude that the unknown is a rod-shaped, Gram positive bacterium (Figure 3A). With this, it was possible to eliminate all Citrobacter, Enterobacter, Escherichia, Proteus, Salmonella, Shigella, Serratia, Klebsiella and Morganella species (as it can be observed in Figure 1). The next step was to perform a catalase and oxidase test on the sample, these tests were chosen because they can eliminate a wide variety of genera and/or species. The results showed that the bacterium was positive for both catalase and oxidase (Figure 4), which narrowed down the identity of the unknown to either B. subtilis or B. laterosporus (as it can be observed in Figure Figure 4: Positive result for the oxidase test 1). The sucrose test was chosen to distinguish between both species of Bacillus and as it can be observed in Figure 5, it gave a negative result, meaning that our unknown sample A is, presumably, B. laterosporus. To confirm the identity of sample A, the BEA, nitrate and MR tests were performed. A negative result was obtained for the BEA test ( Figure 6C) and positives for both the MR and nitrate tests ( Figure 6A & 6B), which match what Figure 5: Negative result for the sucrose test is known about B. laterosporus. A B C All test results for sample A are also summarized in Table 2. Figure 6: A) Positive result for nitrate test; B) Positive result for the MR test; C) Negative result for the BEA test Sample B Initially, sample B was isolated from a mixed culture and then streaked onto a TSA plate. The B A colonies of the sample are round & smooth and present a yellowish Figure 7: A) Gram stain of unknown; B) Colony morphology of sample B. color, which can be observed in Figure 7B. Afterwards, by both visualizing the sample under the microscope and by performing a Gram stain, it was possible to conclude that the unknown is a rod-shaped, Gram negative bacterium (Figure 7A). With this, it was possible to eliminate all Bacillus, Enterococcus, Aerococcus, Lactobacillus, Staphylococcus and Streptococcus species (as it can be observed in Figure 2). The next step was to perform a catalase and oxidase test on the sample, these tests were chosen because they would eliminate a wide variety of genera and/or species. The results showed that the bacterium was positive for catalase but negative for the presence of oxidase, which narrowed down our unknown to one of these options: E. aerogenes, E. coli, P. mirabilis, P. vulgaris, S. choleraesuis, S. flexneri, S. sonnei, S. marescens, K. pneumoniae or M. morganii (as it can be observed in Figure 2). Lactose and sucrose were chosen as the next tests because they eliminated the most options, both tests gave negative results, which can be observed in Figures 8A & 8B. A B The remaining options for our unknown are: P. mirabilis, S. choleraesuis, S. flexneri, S. sonnei, M. morganii. Figure 8: A) Negative result for sucrose test; B) Negative result for lactose test. From this point on, every selective test would eliminate at the most one or two options, so the next assays that were chosen were the urease and citrate tests since they gave results in 24 hours (as opposed to sugar tests which could take up to 48 hours). As it can be observed on Figure 9, urease gave a negative result, while citrate came out positive (no Figure available), this narrowed down the identity of our unknown to, presumably, S. choleraesuis. To confirm the identity of the unknown, mannitol and KIA tests were performed, which gave out positive results for both as it can be seen help to confirm the identity of our bacterium since S. Figure 9: Negative result for urease on Figure 10. Both tests A choleraesuis can produce H2S and can ferment mannitol. B Figure 10: A) Positive result for KIA; B) Positive result for mannitol On Table 2 we can read the condensed test results for both samples A & B. Table 2: Condensed results for both samples Samples Sample A B.laterosporus Sample B S. choleraesuis Tests Results Results Gram stain Positive Negative Catalase test Positive Positive Oxidase test Positive Negative Lactose test Not performed Negative Sucrose test Negative Negative Urease Not performed Negative KIA Not performed Mannitol Not performed BEA test Negative Positive Positive Not performed Nitrate test Positive Not performed MR test Positive Not performed DISCUSSION AND CONCLUSIONS The physiological and biochemical tests used to correctly characterize and identify unknown samples A & B were done without any complications and helped to establish that the samples were B. laterosporus and S. choleraesuis (respectively). The only difficulty that was encountered in this experiment was trying to get a pure isolate of sample A, initial isolation attempts did not work because of an inadequate streaking technique, after this was amended, it was possible to get an isolate that allowed the experiment to continue. S. choleraesuis is a host-adapted pathogen that causes swine paratyphoid and that can be pathogenic to humans ( Wilcock, Schwartz 1992), potentially being able to cause septicemic disease (Blaser, Feldman 1981). Swine are the usual reservoir for this specie, which is a significant concern, not only because of its capacity to induce disease in piglets, but because of the public health implications for humans that consume the meat and might acquire food-borne infections (Chiu et al 2002). The most serious complication of an infection by S. choleraesuis is the development of a mycotic aneurysm, which could be fatal. The recent emergence of strains that are resistant to ampicillin, chloramphenicol, trimethoprim-sulfamethoxazole and fluoroquinolone antibiotics is currently a concern that requires further study (Chiu et al 2004). Furthermore, according to our test results, this bacterium can ferment glucose, citrate and mannitol, produce H 2S and express the catalase enzyme. Bacillus laterosporus, now named Brevibacillus laterosporus (Shida et al 1996), is an aerobic sporeforming bacterium found in water, soil and insects. It has a characteristic ability to produce crystals with potential bioinsecticide properties reported against snails, nematodes and insects (Ruiu 2013). In addition, some strains of B. laterosporus have been reported as being able to produce broad-spectrum antimicrobials with activity against phytopathogenic bacteria and fungi (Chandel et al 2010). The fact that the bacterium can produce both biopesticides and antimicrobials makes B. laterosporus an ideal candidate for agricultural biotechnological applications. According to our test results, this bacterium can ferment glucose, reduce nitrate and can express both the catalase and oxidase enzymes.