Identification and Development of Proteus Mirabilis

The Identification and Development of Proteus mirabilis Using RapID SS/u and Other Biochemical Tests

Abstract

The purpose of this experiment is to comprehend and develop an understanding of the biochemical and physiological purposes of different species of bacteria. To further this concept, the identification of the unknown bacteria #100 is capable through the interpretation of several biochemical and physical test methods. Several methods related to each other and significantly played a key role in the identification process, such as; a positive citrate test, a positive methyl red test and a negative Voges-Prokauer test. These results could be demonstrated in a flowchart, where several other known organisms could be ruled out and the unknown #100 could be determined to be Proteus mirabilis. To further confirm this conclusion, remaining biochemical tests were compared to previously expected data in table 4.1 and was found to be a very accurate representation of the bacteria. A RapID SS/u test was used in attempt to further confirm the identification of unknown #100. It presented positives for Urea (URE), Amino acid bnaphthylamide (A2) and Amino acid b-naphthylamide (A3). The unknown sample didn't align perfectly with any positive results on the differential chart, however it did correlate with the URE and A2 results for Proteus mirabilis. This was not the ideal outcome, but Proteus mirabilis still deemed to be the best identification for the unknown #100. Overall, Identification practices both biochemical and RapID SS/u, were studied and produced the same results for a conclusion to be drawn.

Introduction

The identification of unknown organisms established on indication and biochemical tests is very important for many fields, specifically the medical industry. A collection of assessments that are used for biochemical identification are motility, gram-stain, indole, aero-tolerance, sulfur reduction, methyl red, kligler's iron agar, voges-proskauer, glucose/lactose fermentation and citrate. Species are investigated, and often can be isolated by characteristics such as response to disease outbreaks, identification of toxins, or susceptibility to reactions (Baron, 1996). Microbial identification techniques are being improved or discovered every day, resulting in easier recognition of species or discovering new species all together (Baron, 1996). These types of indication tests specifically are essential when looking to pinpoint the species known as Escherichia coli and Enterobacter aerogene (Hermraj, Diksha, & Avneet, 2013). It is significant to note that these tests used to identify Enterobacteriaceae do not often correlate with the expected results (Holt et al., 1994). Preferably, a test should produce a confident positive or negative result that is accurate. This is not the case and there is currently no biochemical test that has the ability to do so. According to Bergey's manual (1994), the micro-organism being tested is said to be positive if 90% of the results out of 100 applications come back one particular result, either positive or negative. The purpose of this lab is to identify the unknown bacteria #100 using numerous biochemical tests and compare the results to expected data.

The gram staining method is commonly used a grouping procedure, and allows for organisms to be congregated based on their cell walls (Buck, 1982). It's often used as a primary identification tactic throughout many fields as its rapid and relatively easy to comprehend (Buck, 1982). The bacteria that retain the purple/violet colour from the initial steps of the method are known as gram-positive (Coico, 2005). These recognized bacteria have a thick, impermeable cell wall which opposes the decolourization that is forced upon it by alcohol washes. The cell wall is made up of peptidoglycan layers and polymers. The bacteria that appear pink/red after a wash from alcohol are acknowledged as gram-negative. This occurs because they have a thin peptidoglycan layer, which can be influenced by the alcohol wash and produce a pink colour (Coico, 2005).

Motility in bacteria is due to the motion produced by flagella, which are placed in specific orientations on the organism (Conrad, 2012). Being motile is significant to the characteristics of an organism, as motility allows for movement from different environments, and competition with other micro-organisms. Bacteria that lack flagella are still able to move, but through gliding which is a movement across surfaces through the use of biofilms and hydrodynamic interactions (Conrad, 2012). Triphenyltetrazolium chloride (TTC) is a medium used to identify motile organisms, as it is colourless in its oxidized form (Ball & Sellers, 1966). When bacteria are set to grow in TTC, dye is absorbed and reduced to an insoluble red colour which is easily seen when motility occurs. Large areas of red dye can be seen developing away from the line of inoculation, indicating the ability of movement from the organism (Ball & Sellers, 1966).

Based on the response to oxygen, bacteria can be classified as anaerobic, aerobic or facultative (Unden, Becker, Bongaerts, Schirawski, & Six, 1994). Aerobes need and strive in the presence of oxygen, anaerobes are not capable of growth in the presence of oxygen, while facultative organism are adaptable and can develop with or without oxygen (Unden, Becker, Bongaerts, Schirawski, & Six, 1994). This experiment uses the oxygen requirements as an essential for identifying the unknown #100 organism during the classification procedure. Indole production is an intracellular signalling mechanism used by over 85 known species, and is achieved by enzymatic degradation of tryptophan (Hiep Han, Lee, Cho, Wood, & Lee, 2011). Indole production is important in microbial communities, especially the human immune system. As a characteristic, indole production plays a big role in classification as it is a significant phenotypic identifier (Hiep Han, Lee, Cho, Wood, & Lee, 2011). The most commonly used method is the Kovac's assay, which isolates indole producers with a reagent consisting of dimethylaminobenzaldehyde and hydrochloric acid (Pinero-Fernandez, Chimerel, Keyser, & Summers, 2011). A positive result of this test shows red colouration from rosindole dye. A negative result sees no colour change (Pinero-Fernandez, Chimerel, Keyser, & Summers, 2011).

Selected bacteria are capable of reducing sulfate into H2S and use it as the electron acceptor when degrading organic compounds (Muyzer & Stams, 2008). They can thrive in an assortment of environments, while being a mass importance to such ecosystems, as they are responsible for mineralization (Muyzer & Stams, 2008). With the use of a SIM medium, the ability for an organism to reduce sulfate is seen through the production of black precipitation (Holt et al., 1994). A negative result would be no formation of black precipitate (Holt et al., 1994).

The remaining indicator tests had unique purposes that help conclude results in finding the identification of unknown organism #100. The significance behind the KIA test was to inquire about the capability to ferment glucose and lactose, which then produces hydrogen sulfide (Holt et al., 1994). A KIA slant is used to characterise the findings. A red slant indicates glucose fermentation has occurred and a yellow slant indicates lactose fermentation has occurred. The presence of black precipitation is also an indicator that acid was produced (Holt et al., 1994).

The methyl red test is used to signify if an organism produces acids (McDevitt, 2009). A red colour indicates a positive result, where as an orange/yellow colour indicates a negative or inconclusive result. Both negative and positive results can be recorded after a pH indicator has been added to the existing medium. An additional useful test is the Voges-Proskauer. This test considers the organisms capability to take glucose and produce acetylmethylcarbinol from it.

These two tests are usually paired together to characterise groups of endobacteriaceae and actinobacteria. They are also used in unification with indole and citrate tests to make up IMViC chemical testings, which are also used to identify endobacteriaceae (McDevitt, 2009). Finally, the citrate test is used among enteric bacteria to inquire on their ability to use citrate as their lone carbon source (Hermraj, Diksha, & Avneet, 2013). The use of citrate as a carbon source is only fulfilled if the tested organism contains the enzyme citrate permease. A blue colour, and sometimes growth identifies a positive result of citrate utilization. No change indicates a negative result (Hermraj, Diksha, & Avneet, 2013).

Becoming a better method for identifying unknown organism, the RapID SS/u system is a non-growth-dependent micro-method that assists in identifying unknown gram-negative bacilli, gram-positive cocci and other yeasts in a short two hour span (DeGirolmai, Poliferno, Mills, & Eichelberger, 1988). It does so by detecting enzymes that act on chemogenic substrates. The RapID SS/u system is capable of accurately identifying 160 genus's out of 185 isolates, making it much more precise then the several biochemical tests done in lab. Particularly in this experiment, results were viewed in tubes that produced either a positive or negative out of 100 attempts (Carpenter-Cleland, 2019). A negative was considered anything below 70, whereas a positive was considered anything above (Carpenter-Cleland, 2019). When compared to the differential chart, results should be close enough to correctly isolate the organism.

The five organisms that the unknown #100 could possible be are Proteus mirabilis, Escherichia coli, Enterobacter aerogenes, Citrobacter freundoo, and Serrotia marcescens. The anticipated test results can be located in table 4.2. It's important to observe and interpret that all organisms are considered gram-negative, facultative anaerobes.

Materials and Methods

Refer to laboratory 4 manual (Carpenter-Cleland, 2019).

Note there was a change to Procedure 11: We did not perform this procedure and results were not collected.

Note there was a change to Procedure 10: We did a quadrant streak, not a stab and streak inoculation.

Results

Throughout the experiment, several biochemical tests were completed in order to determine numerous characteristics of our unknown intestinal organism (Table 4.3). Beginning with procedure 1, the gram stain procedure was demonstrated and the organism was viewed to be bacilli and red/pink in colour. Procedure 2 tested for motility by showing growth not only on the stab line in the given agar, but spread from the stab line, showing that the organism is motile and has flagellum. During procedure 3 when the organism was fixed to develop on TSA, a general growth of round, entire raised culture was recorded. The culture was also a light beige/brown colour. In addition, growth on other selective medias in procedure 5 such as Endo Agar and MacConkey Agar did not occur, or only occurred in small amounts. To resume, there were only visible traces of growth in FMT inoculated with the unknown organism at the surface of the test tube indicating a obligate aerobe. Indicator tests, such as the citrate test and methyl red test both tested positive, the citrate growth turning blue and the methyl red test medium remaining red. Other indicator tests, such as the indole and the Voges-Proskauer test concluded to be negative as the results for the VP test were yellow, and the indole was white. The KIA test for both lactose fermentation and glucose fermentation resulted in a red slant, yellow along with black precipitate. When compared to table 4.1, the positives and negatives correlate correctly with the bacteria Proteus mirabilis.

Different tests give rise to altering characteristics of the given organism. Results are able to reveal and assist in identifying a specific bacteria. All tests were taken place in the in appropriate media and parameters (see Lab 4 manual).

Table 4.3. Results of Multiple Biochemical and Staining Tests Based on Unknown Organism.

Different tests give rise to altering characteristics of the given organism. Results are able to reveal and assist in identifying a specific bacteria. All tests were taken place in the in appropriate media and parameters (see Lab 4 manual).

Figure 1. Identification Flowchart for Unknown Organism

Illustrates determination of unknown bacterial organism through several biochemical indicator and reagent tests. The unknown organism was concluded to be Proteus mirabilis.

Identification Flow Chart

Highlighted in the red are outlining the results of unknown #100, beginning with the clarity of it being gram negative. Then, all organisms were confirmed to be facultative anaerobes. From there and further down the flow chart, it was seen how the citrate test, methyl red test, VP test and glucose fermentation test were able to differentiate each organism.

RapID SS/u Results

During the RapID SS/u procedure, several reaction cavities came back with positive results in the form of colour changes. For our unknown sample #100, urea was the first to achieve a positive, by presenting a dark red/pink colour (Figure 2). Amino acid b-naphthylamide (A2 & A3) was next to attain a positive, both showing purple/pink results (Figure 2). These positive tests could be compared to the corresponding differential chart to determine the precise identification of out known bacteria results (Carpenter-Cleland, 2019). The bacteria's that closely represented the positive results when compared were Citrobacter spp., Enterobacter spp., Klebsiella spp., Morganella morganii, Proteus spp., Providencia spp., Serratia spp. No known organism displayed positive results for the 3 same tests that also displayed positives in our unknown sample. Because of this, Proteus spp. was found to be the unknown, as both its URE and A2 tests were nearly 100% and this can relate to, and confirm the results from the biochemical tests.

Discussion

After completing the lab and recording the results, it was relevant to compare the results (Table 4.3) to those of the previously known intestinal organisms (Table 4.1). It could be concluded that unknown #100 is Proteus mirabilis, based on the found chemical and physiological experimental results. Theoretically, the RapID SS/u test is a much more accurate way to identify organisms and should be, compared to traditional other biochemical tests, however the RapID SS/u didn't give a clear indication of an organism for this trial (DeGirolmai, Poliferno, Mills, & Eichelberger, 1988). Ideally, the unknown #100 would compare closely with the expected results of another organism on the Differential Chart, but unfortunately it only compared with 2/3 positives for all possible outcomes (Carpenter-Cleland, 2019).

After consideration of the RapID SS/u and the other indicating biochemical tests, it could be concluded that Proteus mirabilis best fit all the results.

The first procedure in the lab was the gram-stain method. The unknown organism #100 was found to be gram-negative, as the cells appeared to be a dark purple/violet colour. This result was not important to the identification of the unknown because all possible outcomes were gram-negative so it didn't provide any differing information (Figure 1). In addition, the aerotolerance test investigated the response to oxygen, providing answers to how much oxygen the organism needs to survive. There was growth throughout the FMT tube by the unknown #100 with a significant amount growing at the surface, indicating a facultative anaerobe. This was not significant in the indications of the unknown because all of the possible organisms were expected to be facultative anaerobes (Figure 1).

The next test that assisted in the Proteus mirabilis indication was the citrate test. The unknown was added to the medium and it resulted in a colour change from green to blue. This signified a positive test result. When compared to the expected characteristics of Escherichia coli, Enterobacter aerogenes, Citrobacter freundii, Proteus mirabilis and Serratia marcescens, all were positive as well besides Escherichia coli (Table 4.1). This dictated that unknown #100 is not Escherichia coli and more likely one of the remaining 4 (Figure 1).

The next test represented on the flowchart (Figure 1) was the methyl red test. When unknown #100 was added into the MRVP medium, a colour change of bright red was viewed. This indicates a positive test result. Of Enterobacter aerogenes, Citrobacter freundii, Proteus mirabilis and Serratia marcescens, Citrobacter freundii andProteus mirabilis are organisms that also produce stable acids and are therefore positive. This dictates that the unknown #100 cannot be Enterobacter aerogenes or Serratia marcescens, as they resulted in negative results. At this point, the unknown can either be Citrobacter freundii or Proteus mirabilis.

To further the results of a negative methyl red test, the flow chart represented the classification achieved by glucose fermentation testing (Figure 1). A positive glucose fermentation test resulted in a red agar slant, not to be confused with lactose fermenting which results in a yellow slant (Table 4.2).

Enterobacter aerogenes is expected to remain red and ferment glucose, concluding in a positive result in contrary to Serratia marcescens (Figure 4.2). This investigation into glucose fermentation did not directly impact the clarification of unknown #100 being Proteus mirabilis because it is classifying 2 organisms that were previously ruled out.

The last biochemical test considered on the flowchart (Figure 1) was the Voges-Proskauer test which was the final step in identifying unknown #100. Once unknown was in the MRVP medium, no change occurred so the results concluded to be negative. The known characteristics of the final two bacteria were positive for Citrobacter freundii and negative for Proteus mirabilis (Table 4.1). Because the unknown was ruled negative, it allows for a final determination of unknown #100 being Proteus mirabilis. After this step of thee flow chart, all 5 known organism have been ruled out to a final end and classified individually (Figure 1).

The indicated tests were all used to signify biochemical and physiological characteristics of Proteus mirabilis, and allow unknown #100 to be identified accordingly (Figure 1). There were several other tests executed, but did not directly interfere with this particular identification process. These tests were Indole, lactose fermentation, sulfur reduction, motility and gelatin hydrolysis. There were also cultural and colonial characteristic recorded that could be compared to those of the known bacteria. Both the cell length, cell shape and colonial characteristic of Proteus mirabilis all align with the views of unknown #100 (Table 4.3), clarifying that it is indeed this organism (Table 4.1).

In comparison to modern day studies, an experiment was conducted in Hyderabad, India, where bacteria isolated from ice cream samples was characterized through the use of several biochemical tests, most of which were also used in this lab (Badr, 2018). The following tests; Indole, methyl red, VogesProkauer, citrate utilization, catalase, oxidase, gelatin hydrolysis, rapid urease and hydrogen sulfide were used to conclude that most ice creams were kept unsterile and in poor condition. A total of 25 species were identified from the collected samples, all could be identified and categorized into 5 separate bacteria species consisting of Escherichia coli, Enterbacter, Klebsiella, Proteus, and staphylococcus. A lot of these species have the potential to damage human health, so it is interesting to see how poorly these samples were kept considered they were supposed to be consumed by humans (Badr, 2018). This study is relevant to the experiment performed because it took samples with numerous unknowns, and thoroughly investigated the species using physical and biochemical tests. The main dissimilarity is that the study conducted with the ice cream samples validates how many organisms can be identified within precise contaminated areas. The experiment performed during Lab 4 was an introduction to accurately identifying organism based on biochemical tests. In comparison to labs that normally perform isolations and identifications in contaminated areas, lab 4 was conducted on smaller and tightly controlled scale.

References

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Appendix

Table 4.1. Physiological and biochemical characteristics of different bacteria.

This table provides the characteristics of 5 known intestinal bacteria. These characteristics are used several times throughout the lab to compare expected results in order to better determine what unknown #100 is.

Table 4.2. Biochemical Tests used in Lab 4.

This table demonstrates the indication of a positive and negative test result dependant on the actual test being considered. It was used to compare the results of the given methods in the lab to properly interpret the conclusions (Holt et al., 1994).

Figure 2. RapID SS/u Report Form for Unknown Organism #100. Displays the several test results based on reaction cavities. Bacterial cultures were incubated at 35-37°C for 4 hours, where colour changes revealed positives for URE, A2 and A3.