Microbiology Lab Report: Isolation & Identification of B. subtilis & P. mirabilis, Lab Reports of Bacteriology

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Microbiology 205

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Major Unknown Report

Suzanne Ricca - Lab #

Gram (+) Unknown #13 – Bacillus subtilis

Gram (-) Unknown #13 – Proteus mirabilis

Isolation and Identification of Gram (+) Organism #13 – Bacillus subtilis

4.16.2012 – Unknown organism was streaked from a mixed broth onto both TSA and BHA plates in an attempt to isolate pure colonies for testing.

4.18.2012 – Although growth was best on the TSA plate, the Gram(+) organism was unable to be isolated because of overgrowth of the Gram(-) organism. The plate showed areas of tiny pinpoint colonies surrounded by cloudy areas, indicating the presence of the Gram(-) organism throughout the plate. This was confirmed with gram staining of the pinpoint colonies which showed the presence of both organisms. Another streak was done on a PEA plate in an attempt to inhibit the growth of the Gram(-) organism enough to isolate the Gram(+).

4.23.2012 – One large circular colony grew on the PEA plate. Gram staining indicated the presence of long pinkish rods with circular purple spots inside the cells. The presence of a faint smaller shape throughout the slide was also noticed, and thought to be spores. Spore staining was inconclusive and the PEA plate was returned to the incubator for further aging. Two TSA slants were inoculated to produce working samples, as well as a motility deep and FTM broth.

4.25.2012 – Gram staining of the TSA working slant showed the same long pinkish rods with purple dots as well as faint shapes in the background. The FTM showed a facultative anaerobe and the motility test showed the organism to be motile. Another spore stain of the PEA plate remained inconclusive and the plate was returned to the incubator. In the likelihood the organism was in fact a spore former and therefore in the Bacillus genus, a mannitol broth was inoculated to begin to differentiate among the Bacillus species.

4.30.2012 – A final spore stain was done on samples taken from the PEA plate as well as the working slant. Although there was no change to the stain from the PEA plate, fortunately the sample from the working slant produced the appearance of spores and confirmed the genus Bacillus. Unfortunately the faint background shapes were not the actual spores but short rod-shaped cells and indicated the continuing presence of the Gram(-) organism. Since the motility test and FTM test were done with the organisms still mixed, they are not able to be used. The mannitol test (read on 4.26) appeared negative, indicating neither organism is a mannitol fermenter, so that test is still good. A sample from the working slant was streaked onto another PEA plate to try to further inhibit the Gram(-) growth and isolate the Bacillus.

5.02.2012 - There was heavy growth throughout the new PEA plate with a few tiny pinpoint colonies but they were not isolated form the rest of the growth. It did not appear as if the second organism was still present however and this was confirmed by doing a gram stain from the growth at the end of the streak. The long pinkish rods with purple spots inside the cells were still present, but no smaller rods were present and the Bacillus seemed to finally be isolated. Two new slants were inoculated to produce new working samples. Since the mannitol test was already done and negative, the species B. subtilis is eliminated. A MRVP broth was inoculated to differentiate between B. megatarium (VP-), B. sphaericus (VP-), B. cereus (VP+) and B. thuringiensis (VP+).

Blood Agar Hemolysis Streak unknown between given samples of B. cereus and B. thuringiensis to compare strength of hemolysis

Growth inconsistent with either species as unknown exhibited unique contoured wrinkles, contamination of mannitol test suspected Mannitol broth (Mannitol fermentation)

Retest mannitol fermentation to differentiate VP positive species B. subtilis and B. megatarium

Weak Positive (yellow-orange with some red remaining), confirms Bacillus subtilis

Habitat & Lifestyle Bacillus subtilis

Generally considered a ubiquitous organism, B. subtilis is found diversely in the soil, plant roots and even the GI tract of animals. It is gram positive and motile by peritrichous flagella. Since it is a spore- former it is able to assume a vegetative state under harsh conditions such as temperature variations and lack of nutrients, and is most often found in this state. It can appear to be an obligate aerobe but more recently it has been observed to grow anaerobically in the presence of nitrate, making it a facultative anaerobe. This supports the idea that B. subtilis can actually germinate within the GI tract of animals, where it has a beneficial probiotic effect that was previously attributed to an unknown property of the spore.

Special Characteristics of Bacillus subtilis

When biologically active, B. subtilis also produces a variety of enzymes which make it an important contributor to nutrient cycling in the soil. It is a promoter of plant growth as well, because it grows in close association with plant roots where it forms beneficial biofilms. It seems B. subtilis has a mutually beneficial symbiotic relationship with the plant rhizosphere; the presence of the microbe prevents the plant from being colonized by other bacteria that would be harmful to it, and the microbe is able to have a place to grow its biofilm and access nutrients.

Clinical Significance of Bacillus subtilis

Although it can be associated with food contamination and occasionally food poisoning, B. subtilis is otherwise non-pathogenic to humans. It exhibits minimal if any virulence, confirmed by genome sequencing of a strain which showed no genes encoding for virulence factors. It is generally not an animal or plant pathogen either, but is actually hazardous to other microorganisms. Genome sequencing has also revealed B. subtilis has a large portion of its genes dedicated to producing the many compounds which inhibit other bacteria and fungi. This feature is what probably enables it to compete in nature and allows it to promote plant growth and act as a probiotic. B. subtilis is used to produce many antibiotics including the commonly known bacitracin, and its enzymes are used for industrial purposes such as protease for detergents. It is also used to produce antifungal agents for plants in agricultural settings.

Bacillus subtilis Identification Flow Chart

Gram (+)

Cocci Bacilli

Spore Former

Bacillus

Mannitol Fermentation (-)

B. sphaericus, B. cereus, B.

thuringiensis,

B. megatarium (V)

VP (+) B. cereus B. thuringiensis

Hemolytic Activity

VP (-) B. megatarium B. sphaericus

Mannitol Fermentation (+) B. subtilus B. megatarium (V)

VP (+) B. subtilis

Bacillus subtilis

VP (-) B. megatarium

Non-Spore Former

Media Table

Test Purpose Result

Gram stain Determine morphology and arrangement

Gram negative, short red rods (bacilli) FTM broth Determine oxygen requirements to differentiate bacilli between aerobes and facultative anaerobes

Facultative anaerobe (growth throughout)

Motility Deep Differentiate facultative anaerobes between motile and non-motile

Motile (growth spread throughout) indicates organism is an enteric Lactose broth Determine lactose fermentation to differentiate enterics between fermenters and non-fermenters

Negative (red) indicates non- lactose fermenter

Simmon’s Citrate (Citrase production)

Determine ability to use citrate as carbon source to differentiate non-lactose fermenting enterics

Positive (blue) indicates Salmonella , Serratia or Proteus

MRVP Use VP test to determine acetoin production and differentiate between Salmonella , Serratia and Proteus

VP negative (amber/green) indicates Salmonella or Proteus

Urease Determine ability to metabolize urea and differentiate between Salmonella and Proteus

Positive (bright pink) indicates Proteus mirabilis or vulgaris

Ornithine Decarboxylase Determine ability to decarboxylate ornithine to putrescine and differentiate between P. mirabilis and P. vulgaris

Positive (purple) indicates P. mirabilis

Tryptone Determine ability to produce indole and confirm differentiation between P. mirabilis and P. vulgaris

Negative (no color change) confirms Proteus mirabilis

Habitat & Lifestyle of Proteus mirabilis

As a member of the Enterobacteriaceae Family, P. mirabilis is a gram-negative facultative anaerobe and motile by peritrichous flagella which contributes to its swarming ability. It is among the normal flora of the human intestine and also found in the urinary tract. In nature it can be found freely living in water and moist soil. P. mirabilis survives best in an alkaline environment, with a pH between 8-9.

Special Characteristics of Proteus mirabilis

A unique feature of P. mirabilis is the ability to differentiate its morphology between swimmer cells and swarmer cells. Swimmer cells, very small flagellated rods, are found in liquid suspension. Conversion to swarmer cells, in which the cell becomes elongated with highly flagellated filaments, occurs on sold

surfaces and the cells line up and move in raft formation. On growth media such as agar, P. mirabilis has the ability to produce geometric shapes as it grows, called concentric patterns. This is caused by repeating swarming-plus-consolidation cycles in which the organism switches between its swimmer and swarmer state, forming circular terraces. The swarming ability is important in the organism’s virulence.

P. mirabilis raft formation P. mirabilis concentric growth on agar

Clinical Significance of Proteus mirabilis

In a normally functioning urinary tract, P. mirabilis is of little risk and is flushed out regularly before it colonizes. It can become pathogenic and is the cause of complicated urinary tract infections in cases of structural abnormalities of the urinary tract, in immune-compromised individuals, or most commonly in cases of long-term catheterization. P. mirabilis is able to adhere to the catheter and form a biofilm. Due to its motility it can travel up the urethra to the bladder. It also possesses various kinds of fimbriae which enhances its ability to adhere to the host tissue. Once established the ability to produce urease further enhances virulence, allowing P. mirabilis to efficiently metabolize urea into ammonia and carbon dioxide. This increases pH of the area to the microbe’s preferred alkaline level, as well as causes bladder and kidney stones, which it is also able to colonize for use as further protection from host defenses and antimicrobial drugs. In addition, P. mirabilis can evade host defenses by producing protease which degrades the IgA released by the host, as well as releasing its endotoxin hemolysin which is cytotoxic to the host epithelial cells.

Although other Proteus species have become resistant to many antibiotics, P. mirabilis remains susceptible to most except tetracycline. Infections caused by P. mirabilis can most likely be treated with ampicillin and other broad spectrum penicillins, cephalosporins, imipenem, and aztreonam, but is still difficult to clear with antibiotics alone. This is due to the stone formation which can become reservoirs for the infection to reoccur, eventually travelling to the kidneys and causing further complications including pyelonephritis and renal damage. Even more serious, the microbe can cause bacteremia by invading the bloodstream where its endotoxin can induce sepsis, resulting in Systemic Inflammatory Response Syndrome (SIRS) which can be fatal. Research is also currently in process to develop a vaccine involving the MR/P (mannose-resistant Proteus -like) fimbriae, one of at least four types of known fimbriae produced by P. mirabilis and known to function as a surface antigen. The vaccine would not only have the potential to prevent complicated urinary tract infections from Proteus in high-risk individuals but from other microbes as well which cause similar conditions.

Works Cited

Basic Characteristics for Identification of Selected Bacillus Species http://faculty.sdmiramar.edu/dtrubovitz/micro/bacillustable.pdf

Bacillus cereus vs Bacillus thuringiensis http://faculty.sdmiramar.edu/dtrubovitz/micro/BCversusBT.pdf

“Bacillus subtilis Final Risk Assessment.” US Environmental Protection Agency, Feb 1997. http://www.epa.gov/oppt/biotech/pubs/fra/fra009.htm

Earl, Ashlee M., Richard Losick, and Roberto Kolter. "Ecology and Genomics of Bacillus subtilis ." Trends In Microbiology 16.6 (2008): 269-75. Harvard University Department of Molecular and Cellular Biology. Harvard University, 2008. Web. http://www.mcb.harvard.edu/Losick/Pubs/documents/Earl%20and%20Losick%20Ecology%20and%20ge nomics.pdf

Differentiation of Enterobacteriaceae by Biochemical Tests. Difco Manual Tenth edition page 836. http://faculty.sdmiramar.edu/dtrubovitz/micro/Enterobacteriaceae.pdf

“Proteus mirabillis .” BioMed healthcare Technology Cooperative. 2011 http://www.biomedhtc.org.uk/ProteusMirabilis.htm

" Proteus mirabilis : Model for Pathogenesis in the Urinary Tract." Mobley Research Laboratory. University of Michigan Medical School. 29 Nov 2008 http://www.umich.edu/~hltmlab/research/mirabillis/model.htm

Pearson, Melanie. "Complete genome sequence of uropathogenic Proteus mirabilis , a master of both adherence and motility." Journal of Bacteriology 190.11(2008): 4027-37. National Center for Biotechnology Information. US National Library of Medicine. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2395036/?tool=pubmed

Rauprich, Oliver. "Periodic Phenomena in Proteus Mirabilis Swarm Colony Development." Journal of Bacteriology 178.22 (1996): 6525-538. National Center for Biotechnology Information. US National Library of Medicine. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC178539/pdf/1786525.pdf

Struble, Kelley. “ Proteus Infections”. Updated 18 Aug 2011. http://emedicine.medscape.com/article/226434-overview