Microbiology 205. Major Unknown Report. Suzanne Ricca - Lab #22. Gram (+) Unknown #13 Bacillus subtilis Gram (-) Unknown #13 Proteus mirabilis

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1 Microbiology 205 Major Unknown Report Suzanne Ricca - Lab #22 Gram (+) Unknown #13 Bacillus subtilis Gram (-) Unknown #13 Proteus mirabilis

2 Isolation and Identification of Gram (+) Organism #13 Bacillus subtilis Unknown organism was streaked from a mixed broth onto both TSA and BHA plates in an attempt to isolate pure colonies for testing 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(+) 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 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 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 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+).

3 VP test was positive. To differentiate between B. cereus and B. thuringiensis a blood agar plate was streaked to determine strength of hemolysis, since the species are similar in almost every other way. They are both hemolytic but B. cereus is stronger. Samples of known B. cereus and B. thuringiensis were also streaked on the plate to compare with the unknown The hemolysis result was not consistent enough with either known species to be definitive. It looked most like B. thuringiensis but the unknown growth had raised wrinkled areas and appeared unique enough to be a separate species. A problem with the mannitol test is suspected and another mannitol broth was inoculated to retest for B. subtilis The mannitol test was not a strong positive, but still clearly positive. The broth turned yellow-orange at the top although remained red toward the bottom and there were a few bubbles in the tube. Most of the growth was at the top indicating the possibility of an aerobe rather than facultative anaerobe. Since the VP test is already done and positive, Bacillus subtilis is confirmed. Mannitol retest (+) VP (+) Hemolysis: Unknown in Middle Media Table Gram stain Spore stain FTM broth Motility Deep PEA Plate Mannitol broth (Mannitol Fermentation) MRVP (Acetoin production) Test Purpose Result Determine morphology and Gram positive, long pinkish rods arrangement (bacilli) with purple dots Differentiate gram positive bacilli Spore former, indicating Bacillus, between spore formers and nonspore also showed gram negative formers organism still present Determine oxygen requirements and differentiate Bacillus species Determine motility and differentiate Bacillus species Inhibit gram negative growth and isolate gram positive Differentiate Bacillus between fermenters and non-fermenters Use VP test to differentiate mannitol non-fermenters Facultative anaerobe (growth throughout), may not be accurate due to gram negative Motile, consistent with all possible Bacillus species Gram positive isolated, confirmed with gram srain Negative (red) indicates B. cereus, B. megatarium, B. sphaericus or B. thuringiensis VP positive (red) indicates B. cereus or B. thuringiensis

4 Blood Agar Hemolysis Mannitol broth (Mannitol fermentation) Streak unknown between given samples of B. cereus and B. thuringiensis to compare strength of hemolysis Retest mannitol fermentation to differentiate VP positive species B. subtilis and B. megatarium Growth inconsistent with either species as unknown exhibited unique contoured wrinkles, contamination of mannitol test suspected 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 sporeformer 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.

5 Bacillus subtilis Identification Flow Chart Gram (+) Cocci Bacilli Spore Former Non-Spore Former Bacillus Mannitol Fermentation (-) B. sphaericus, B. cereus, B. thuringiensis, B. megatarium (V) Mannitol Fermentation (+) B. subtilus B. megatarium (V) VP (+) B. cereus B. thuringiensis VP (-) B. megatarium B. sphaericus VP (+) B. subtilis VP (-) B. megatarium Hemolytic Activity Bacillus subtilis

6 Isolation and Identification of Gram (-) Organism #13 Proteus mirabilis Unknown organism was streaked from a mixed broth onto both TSA and BHA plates in an attempt to isolate pure colonies for testing Although growth was best on the TSA plate, the organism was not isolated into pure colonies because of its swarming growth throughout the entire plate. It grew over and around the colonies of the other organism and appeared as a cloudy area where the other organism did not grow. A sample was taken from the cloudy area, gram stained, and determined to be mostly short and some longer red rods, indicating isolation of the Gram(-) organism. Two slants were inoculated to produce working samples as well as inoculation of a motility deep and incubated The motility test was positive and an FTM broth was inoculated to determine respiration The FTM test indicated a facultative anaerobe. Combining this result with the motility test determines the organism is an enteric. A lactose broth was inoculated to differentiate between the lactose fermenting and non-lactose fermenting genera. Chromobacterium is already eliminated as a possibility because the organism did not have the characteristic violet pigment on the growth media. A Simmon s citrate slant was also inoculated to further differentiate, as the only genus with citrase variability is Proteus The lactose test (read on 4.26) was negative indicating a non-lactose fermenter and eliminating Escherichia, Enterobacter and Citrobacter. The citrate test was positive indicating the organism produces the citrase enzyme to use citrate as its carbon source. This eliminates Morganella and Edwardsiella and narrows the potential genus to Salmonella, Serratia or Proteus. An MRVP broth was inoculated to differentiate Serratia (VP+) from Salmonella (VP-) and Proteus (VP-, although one species can be variable). A urea broth was inoculated to further differentiate Salmonella (urease-) and Proteus (urease+, although one species can be variable) As suspected the organism was VP(-) and urease(+), confirming Proteus as the genus. A tryptone broth to test for indole production and an Orthinine decarboxylase broth were both inoculated to differentiate between P. vulgaris and P. mirabilis Ornithine decarboxylase test was positive and indole test was negative, both consistent for Proteus mirabilis. Left: Urease (+), Right: VP (-) Left: Tryptone (-), Right: Ornithine (+)

7 Media Table Gram stain FTM broth Motility Deep Lactose broth Simmon s Citrate (Citrase production) MRVP Urease Ornithine Decarboxylase Tryptone Test Purpose Result Determine morphology and Gram negative, short red rods arrangement (bacilli) Determine oxygen requirements Facultative anaerobe (growth to differentiate bacilli between throughout) aerobes and facultative anaerobes Differentiate facultative anaerobes between motile and non-motile Determine lactose fermentation to differentiate enterics between fermenters and non-fermenters Determine ability to use citrate as carbon source to differentiate non-lactose fermenting enterics Use VP test to determine acetoin production and differentiate between Salmonella, Serratia and Proteus Determine ability to metabolize urea and differentiate between Salmonella and Proteus Determine ability to decarboxylate ornithine to putrescine and differentiate between P. mirabilis and P. vulgaris Determine ability to produce indole and confirm differentiation between P. mirabilis and P. vulgaris Motile (growth spread throughout) indicates organism is an enteric Negative (red) indicates nonlactose fermenter Positive (blue) indicates Salmonella, Serratia or Proteus VP negative (amber/green) indicates Salmonella or Proteus Positive (bright pink) indicates Proteus mirabilis or vulgaris Positive (purple) indicates P. mirabilis 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

8 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.

9 Proteus mirabilis Identification Flow Chart Gram (-) Cocci Bacilli Facultative Anaerobe Aerobe Motile Lactose Fermentation (-) Proteus, Morganella, Salmonella, Edwardsiella, Serratia Lactose Fermentation (+) Escherichia, Enterobacter, Citrobacter Citrate (-) Morganella Edwardsiella Citrate (+) Salmonella, Serratia, Proteus VP (+) Serratia VP (-) Salmonella, Proteus Urease (+) Proteus Urease (-) Salmonella Indole (+), Orthinine (-) P. vulgaris Indole (-), Orthinine (+) P. mirabilis Proteus mirabilis

10 Works Cited Basic Characteristics for Identification of Selected Bacillus Species Bacillus cereus vs Bacillus thuringiensis Bacillus subtilis Final Risk Assessment. US Environmental Protection Agency, Feb Earl, Ashlee M., Richard Losick, and Roberto Kolter. "Ecology and Genomics of Bacillus subtilis." Trends In Microbiology 16.6 (2008): Harvard University Department of Molecular and Cellular Biology. Harvard University, Web. nomics.pdf Differentiation of Enterobacteriaceae by Biochemical Tests. Difco Manual Tenth edition page Proteus mirabillis. BioMed healthcare Technology Cooperative "Proteus mirabilis: Model for Pathogenesis in the Urinary Tract." Mobley Research Laboratory. University of Michigan Medical School. 29 Nov Pearson, Melanie. "Complete genome sequence of uropathogenic Proteus mirabilis, a master of both adherence and motility." Journal of Bacteriology (2008): National Center for Biotechnology Information. US National Library of Medicine. Rauprich, Oliver. "Periodic Phenomena in Proteus Mirabilis Swarm Colony Development."Journal of Bacteriology (1996): National Center for Biotechnology Information. US National Library of Medicine. Struble, Kelley. Proteus Infections. Updated 18 Aug