Lab report on Unknown Microbe Identification

MICROBIOLOGY

Identification of Unknown Organism

NAME: MUHAMMAD AKHTAR BIN MOHD SOFFIEE STUDENT ID: S3579450 COURSE CODE: BIOL 2158 COURSE NAME: BACHELOR OF SCIENCE (BIOLOGICAL SCIENCES) PRACTICAL GROUP: NO. 4 UNKNOWN ID NO.: C47

Introduction

There are numerous types of bacteria that can be found all around us, each with their own unique phenotypic qualities. Those that are potentially pathogenic, especially towards humans, require very accurate identification, so that appropriate actions can be taken to avoid or treat infections. This subject being of the utmost importance considering at least 210, 000 patients per year die due to misdiagnosis in the US alone (James, 2013). The general approach towards identifying bacteria is to subject the isolated organism through a series of biochemical tests, usually beginning with a Gram stain, and using the reaction results to infer morphology and metabolism characteristics. The objective of the investigation is to identify the unknown organism C47, which was isolated from ventilator-associated pneumonia patient, using acquired knowledge from previous microbiology lab sessions. Cause of the infection might also be inferred.

Materials and Methods Isolated organism on Nutrient Agar incubated aerobically at 35 oC for 24 hours was supplied and utilised for the first set of biochemical tests. Colony morphology was noted, then a Gram stain was conducted using a heat fixed smear of the organism, 1% Crystal Violet solution, Jenson’s Iodine, acetone, and dilute Carbol Fuschin. Gram morphology was determined. Separate colonies of the organism were then tested with 1% Oxidase reagent on filter paper for Oxidase test, and 3% hydrogen peroxide for Catalase test. Once all test results were recorded, the organism was streaked on a split agar plate of MacConkey Agar (MCA) and Horse Blood Agar (HBA), then incubated aerobically at 35 oC for 24 hours (RMIT University, 2017). Subsequent biochemical tests were planned for the second session based on first session results.

In the second lab session, presence of growth on the split agar plate is noted for differentiation. Separate colony samples were taken again, and used to inoculate a Klinger Iron agar slope, Craigie tube, peptone water, Lysine basal medium and control basal medium, on separate occasions. A layer of sterile paraffin oil was also added to Lysine and control basal mediums to create anaerobic conditions. These inoculations were setup for H2S production, motility at 37 oC, Lysine decarboxylation, and Indole tests, in that order. Testing for acid from Lactose could also be obtained from Klinger Iron Agar. Peptone water, Klinger Iron agar slope and Craigie tube were sent for aerobic incubation at 37 oC for 24 hours, while Lysine and control basal mediums were incubated similarly, except for 96 hours (4 days). All biochemical tests were carried out with reference to RMIT laboratory manual (RMIT University, 2017), and all agar constituents were as stated on the Oxoid database (Oxoid.com, 2017). A refined flow chart for identification down to species level was constructed with the aid of RMIT ID tables (RMIT Microbiology Unit, 2017).

On the third and final lab session, the results for H 2S production, motility at 37 oC, Lysine decarboxylation, and acid from Lactose were recorded immediately. Five drops of Kovac’s reagent was added to cultured peptone water, shaken gently, then results were noted for Indole production. Test results were doubly confirmed with reference to G.E.R.M.M. (Grand and Paras, 2009). In correlation with biochemical test results, the flow chart was traced and organism species identity was found.

Results (Part A) Table 1: Outcomes of biochemical tests conducted on C47 unknown organism Criteria for testing Colony morphology on NA

Observation Crenate yellow colonies

Inference Growth, non-fastidious

Pink, rod-like cells

Gram Negative (-) Cocco-bacilli chains

Catalase Test

Gas bubbles produced

Catalase Positive (+)

Oxidase Test

No colour change on filter paper

Oxidase Negative (-)

Growth on MCA

Red colonies

Positive (+) Growth, bile tolerant, lactose fermenting

Growth on HBA

Yellow colonies

Positive (+) Growth

H2S production

Yellow coloration on Klinger Iron agar slope and butt

Negative (-) No H2S production

Motility at 37 oC

Cloudy light-yellow coloration in Craigie tube medium

Positive (+) Motile

Lysine decarboxylase

Purple Lysine, Yellow control basal medium

Positive (+) Lysine Decarboxylation

Indole

No change in meniscus area of media

Negative (-) No cleaving of Indole

Acid from Lactose

Yellow coloration on Klinger Iron agar slope and butt

Positive (+) Utilisation of Lactose

Gram morphology

Results (Part B) The identity of the organism was determined by process of elimination, starting with the results of the Gram stain, which identified it as a Gram Negative bacilli (thin peptidoglycan layer and rod shaped). The initial criteria given us, that the organism was not nutritionally fastidious, eliminated Haemophilus, Pasteurella, Legionella, Campylobacter, Helicobacter, and Bacteroids from being potential suspects. Acinetobacter was also eliminated early on, due to species non-presence in ID tables (RMIT Microbiology Unit, 2017). From there, the oxidase negativity (coliform) of the organism enabled the elimination of Vibrio and Pseudomonas species, leaving only the Enterobacteriaceae family. With the inability for the organism to produce H2S, both Salmonella and Proteus species were eliminated. Since the organism was motile at 37 oC, it can be deduced that it has a means of travel (such as by flagella), hence Klebsiella species could be eliminated. The inability of the organism to cleave indole from tryptophan molecules meant that it could not be Escherichia either. The next means of differentiation was from the ability to utilise lactose sugar as an energy source,

eliminating the possibility of a Seratia species, and confirming that it was an Enterobacter.

Finally, to identify the specific species, the positive result for lysine amino acid decarboxylation was used, and confirmed that the unknown organism was Enterobacter aerogenes. The Shigella, Citrobacter, Morganella and Providencia genera were mistakenly not included within the flow chart, but have been verified to be non-possibilities (RMIT Microbiology Unit, 2017).

Discussion During the process of identification, a single complication was encountered where the catalase test result in the first lab session did not yield the desired result upon further inspection. This was because no Gram negative bacillus, that was also not fastidious, was both catalase and oxidase negative (RMIT Microbiology Unit, 2017). In response, both catalase and oxidase tests were repeated in the second lab session. It was found through multiple attempts of the two tests that the catalase test was the source of the problem, as the organism was catalase positive. It was speculated that either the hydrogen peroxide was faulty during the first lab session, or an insufficient amount of the organism was smeared on the glass slip.

Further tests that could be suggested to further confirm the organism identity would include a carbohydrate fermentation broth for Lactose fermentation. Although lactose fermentation could be concluded from the yellow colouration of slope and butt of Klinger Iron Slope, it is not 100% conclusive, when compared to a fermentation broth. Besides that, an Arginine dihydrolase test might also be advantageous in further confirming Enterobacter species, in supplement to the evidence given in lysine decarboxylation (RMIT Microbiology Unit, 2017).

If Enterobacter aerogenes were to be associated with the scenario, it could have been a contributing factor in the worsening condition of the pneumonia patient towards ventilator reliance, as multiple other strains of the Enterobacteriaceae family have been isolated from other patients with the same condition (Fathy et al., 2013). Alternatively, it could have infected the patient after subsequent hospitalization, as Enterobacter is often an opportunistic nosocomial pathogen, especially in the immune-compromised (RMIT Microbiology Unit, 2017). The significance of this possibility is only enhanced by the fact that E. aerogenes is, in general, one of the most frequently isolated species from infected hospitalized patients (Lederberg and Alexander, 2000). If the organism was not originally from the hospital, the patient likely contracted it through contact with soil, water, decaying vegetation, sewage or even some dairy products as potential sources (RMIT Microbiology Unit, 2017). The methods used in identification of the organism could also be further verified as accurate, due to a similar such method and result being obtained in a scholarly study and characterisation of E. aerogenes (Collins et al., 2004).

In consideration of the conclusive evidence towards E. aerogenes being the organism investigated, it can be inferred that the identification of unknown organism C47 was successful. Since multiple bacteria are usually responsible for ventilator-associated pneumonia, and E. aerogenes is primarily an opportunistic bacterium, it cannot be verified if it had caused the initial infection, even though it contributed to its severity.

References -

Collins, C., Grange, J., Lyne, P. and Falkinham III, J. (2004). Collins and Lyne's Microbiological Methods. 8th ed. London, GBR: CRC Press.

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Fathy, A., Abdelhafeez, R., EL-Gilany, A. and Elhafez, S. (2013). Analysis of ventilator associated pneumonia (VAP) studies in Egyptian University Hospitals.

Egyptian Journal of Chest Diseases and Tuberculosis, 62(1), pp.17-25.

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Grand, O. and Paras, C. (2009). [online] Glossary and Electronic Resources of Microbiology Methods. Available at: https://lms.rmit.edu.au/bbcswebdav/pid8031552-dt-content-rid-18625019_1/courses/BIOL2159_1710/Glossary%20for %20Resources%20in%20Electronic%20Methods%20in%20Microbiology %20%28GERMM%29/eb8718050922c506f8dfe4997ad3e83f.html [Accessed 16 Apr. 2017].

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James, J. (2013). A New, Evidence-based Estimate of Patient Harms Associated with Hospital Care. Journal of Patient Safety, 9(3), pp.122-128.

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Lederberg, J. and Alexander, M. (2000). Encyclopedia of microbiology. 2nd ed. San Diego: Academic P.

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Oxoid.com. (2017). Oxoid - Dedicated to Microbiology. [online] Available at: http://www.oxoid.com/uk/blue/ [Accessed 28 May 2017].

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RMIT Microbiology Unit (2017). Identification Tables for Bacteria. 1st ed.

Melbourne: School of Science RMIT.

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RMIT University, (2017). Microbiology 1 Labratory Manual. 1st ed. Melbourne: RMIT University, pp.55-57.

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Signed, Akhtar Soffiee