This laboratory experiment was conducted with the intent of identifying an unknown bacterium that was found in a patient’s urine sample and to determine if the patient currently was in possession of a UTI. This process involved using a dilution of both the patient’s urine and as a control a student’s urine. This dilution was transferred to both a TSA and a MAC plate. After the transfer, these plates were then left in incubation at 37°C for 24 hours. Once this time frame was completed the colony morphology was recorded for both plates and the patient’s unknown bacterium was tested using a lactose fermentation test, an oxidase test, and an API test. After testing was complete the API test was left to incubate at 37°C overnight, the results produced gave a negative test result for the lactose fermentation and oxidase test, a provided a match for Salmonella spp. The recorded colony morphology in addition to the lactose and oxidase test was further used to confirm the identity of Salmonella spp.
Introduction
The urinary tracts of mammals are an intricate system that has many ways to ward off and prevent microbes from proliferating. This includes high salt concentrations, high levels of urea, organic acids and a high Ph. (Behzadi et al 2010). Although the urinary tract is usually sterile, the urogenital area is susceptible to bacteria that have found ways of surviving this hostile environment. Though it is not an ideal habitat for many bacteria, some bacteria are quite well adapted to adhering and proliferating in this urogenital area, so well adapted in fact that humans have a normal flora of bacteria that reside in this area. For adult women, this includes bacteria such as L. acidophilus, corynebacteria, peptostreptococci, staphylococci, streptococci, and Bacteroides. (Davis 1996) For men the bacteria are different and tend to include; S. epidermis, enterococci, and diptheroids, but can also contain E. Coli, Proteus, and Neisseria. (Davis, 1996). The problem derives from when these bacteria that are normal in the urogenital region start to inhabit and even proliferate in the usually sterile urinary tract system when the tissue of the urinary tract system becomes invaded by microbes it becomes what is called a urinary tract infection. (Behzadi et al 2010). UTI’s are a common bacterial infection in women and affects 50-60% of women over the course of their lives, with the leading cause of infection being frequent sexual intercourse (Al-Badr and Al-Shaikh 2013).
Among the pathogens that typically cause a UTI, most fall into the group of Enterobacteriaceae (Farajina et al, 2009). Since most are found within this group, the API test is commonly used to identify bacteria from urine (d’Entremont, H. Maynard, A. pg. 28-33). In order to perform an API test first the bacteria must be grown in on a Petri dish, since the goal is the eventual identification of the unknown bacteria, it can be grown on multiple types of agar media such as TSA, Blood Agar, and MAC agar in order to obtain additional information regarding the bacteria, such as gram negative or gram positive or the specific growth requirements of the bacteria in question (d’Entremont, H. Maynard, A. pg. 28-33). During this lab experiment, the main objective was to determine if a given student sample or the given patient sample contained a UTI.
Materials and Methods
This experiment was conducted using the procedure and materials from the BIOL fall 2018 laboratory manual (d’Entremont, H. Maynard, A. pg. 28-33)
Results
During the lab, the colony morphology, and test results for lactase as well as for the API tests were recorded. Some key information that helped in the identification of the bacterium was the negative test results for the oxidase test (Table 1.2). Another invaluable piece of information was the CFU/ML calculation, which played a vital role in determining the presence of a UTI in the patient (Table 1.1).
Table 1.1: Colony morphology and CFU/ml of student and patient bacteria grown on TSA and MAC Plates for 24 hours while incubated at 37°C.
TSA plate
Student TSA plate
Patient MAC Plate Student MAC Plate Patient
Colony Surface Smooth, Glistening Smooth, Glistening N/A Smooth, Glistening
Optics Opaque Opaque NA Translucent
Pigment Non-soluble, white Non-soluble, white NA Colorless
Form Circular Circular NA Non-soluble
Elevation Flat Flat NA Flat
Bacterial Counts 1.0*101CFU/ML 6.25*103 CFU/ML NA 3.45*103 CFU/ML
Lactase Test NA NA NA Negative
Table 1.2: API test of patient’s urine bacteria, bacteria were grown and that was incubated
at 37°C for 24 hours, and possessed a CFU/Ml of 3.45 *103
MacConkey’s Plate API Negatives
API Positive Oxidase Results Bacterium ID
Patients Unknown Bacterium ONPG, URE, TDA, IND GLU, ARA, LDC, ODC, CIT, H2S, NO2 Negative Salmonella spp.
Discussion
During the analysis, if the sample of the urine provided by the sample, it was determined that the patient had a UTI. This conclusion was reached by first comparing the CFU/ML to the criteria of a UTI. In this comparison it was found that although the typical guidelines for a UTI is >100,000 colonies per ml of urine, some experts believe that this number is too high and misses a significant portion of UTI’s that fall below the >100,000 per CFU/ml range, with this in mind the recommended range of CFU/Ml is suggested is ranged at >1,000. (Schmiemann et al 2010). Using these guidelines the number of CFU/Ml observed qualifies as a potential UTI.
To identify the unknown bacterium both the results from the API test and the Colony Morphology was used. To ensure that the API testing was a valid match for the bacterium Salmonella spp the results obtained was compared to that of a typical testing of Salmonella spp. The results were found to be consistent with the observed results with the exception of a negative reading found for CIT (Imen et al 2012). Since the test of CIT on Salmonella spp is only positive ~74% of the time, the observed results could still be viable to that of Salmonella spp. In order to further the reliability of the test results, the colony morphology and oxidative tests were compared to that of known Salmonella spp. traits. In the comparison of the colony morphology of Salmonella spp. to that of the observed morphology, it was found that the TSA plate morphology descriptions matched that of the observed morphological descriptions (Anriany et al 2001), this was also the case with the morphologies of the MAC plate (Bergeys, 1957 370-373).
During the lab some sources of error were noted, the first source of error being contamination of the urine sample during the cross-stream extraction was a possibility, this had the potential of adding a separate bacterium to the plates, which could have had an impact on the growth of the bacterium. The second source of error was observed in the preparation of the API strips since a relatively small amount of the sample is inserted into the well, a small amount of contamination could have a significant impact on the overall test results. It was also noted that in addition to the tests performed, conducting both a hemolysis test and a catalase test could have provided further evidence of the bacterium’s identification. With the addition of these two tests, the evidence supporting the argument for the correct identification of the bacterium would be significantly stronger.
Conclusion
During this experiment, the unknown bacterium from a patients sample was grown and tested by API to determine the identity of the Bacterium. The CFU/ML was also determined which helped to indicate if a UTI was indeed present. Since the Identification was carried out successfully and the presence of the UTI was verified, the lab experiment was completed as designed and was a success.
References
- Al-Badr, A. Al-Shaikh, G. 2013. Recurrent Urinary Tract Infections Management in Women A Review. Sultan Qaboos University Medical Journal. 13(3): 359-367
- Anriany, Y.A., Weiner, M. R., Johnson, J,A., De Rezende, C.E., Joseph, S.W. 2001. Salmonella enterica Serovar Typhimurium DT104 Displays a Rugose Phenotype. Journal of Applied and Environmental Microbiology. 67(9): 4048-4056
- Behzadi, P. Behazadi, E. Yazdanbod, H. Aghapour, R. Akbari-Cheshmeh, M. Salehian-Omran, D. 2010. A Survey On Urinary Tract Infections Associated With The Three Most Common Uropathogenic Bacteria. Maedica. 5(2): 111-115.
- Bergey, D.H. 1957. Bergey’s Manual of determinative bacteriology. The Williams & Wilkins Co. Baltimore.
- Davis, C.P. 1996. Normal Flora. Medical Microbiology 4th edition. University of Texas Medical Branch at Galveston.
- Farajnia, S. Alikhani, M.Y. Ghotaslou, R. Naghili, B. Nakhlband, A. 2009. Causative Agents and Antimicrobial Susceptibilities of Urinary Tract Infection in the Northwest of Iran. International Journal of Infectious Diseases. 13(2): 140-144
- Imen, B.S. Ridha, M. Mahjoub, A. 2012. Laboratory Typing Methods for Diagnostic of Salmonella Strains, the “Old” Organism That Continued Challenges. Salmonella – a dangerous pathogen.
- Rowe, T.A. Juthani-Mehta, M. 2013. Diagnosis and management of Urinary Tract Infection in Older Adults. Journal of Infectious Disease Clinics of North America. 28(1): 75- 89
- Schmiemann, G. Kniehl, E. Gebhardt, K. Matejczyk, M.M. Hummers-Pradier, E. 2010. The Diagnosis of Urinary Tract Infection. Journal of Deutsches Ärzteblatt International. 107(21): 361-367