Pseudomonas Putida
Welcome to my Adopt a Microbe page! Here, we will be exploring the qualities and characteristics of a popular microbe, Pseudomonas putida. Enjoy!
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BACKGROUND and treatment
BACKGROUND and treatment
Pseudomonas putida is a gram-negative bacterium normally found in soil and freshwater. It has also been reported as an opportunistic pathogen that can cause infections in humans. It is a low-virulence pathogen. It causes nosocomial infections in immuno-compromised individuals and can also cause bacteremia. For example, P. putida is known to effect people with cystic fibrosis.
People who are immuno-compromised are most susceptible to Pseudomonas putida and most isolated species are found in patients who acquired the infections while in the hospital. Safety measures in the hospital may help to decrease potential infection.
There has been an emergence of strains that have acquired resistance, especially in hospitals where the microbes can easily evolve. These evolved, multi-drug resistance strains cause nosocomial infections that are very difficult to treat, especially since these strains are resistant to common antibiotics like carbapenem. Some antibiotics that have been shown to work in the literature are arbekacin and colistimethate.
visualizing
visualizing
p. putida gram stain compared to other bacteria
p. putida gram stain compared to other bacteria
P. putida are a gram-negative bacteria. In the image on the left, we can see P. putida (d) compared to a gram-positive bacteria, E. faecalis (c).
Figure 6 from Zhang, Xinru & Zhang, Qian & Yan, Tao & Jiang, Zeyi & Zhang, Xinxin & Zuo, Yi. (2014). Surface Free Energy Activated High-Throughput Cell Sorting. Analytical chemistry. 86. 10.1021/ac503100a.Pseudomonas putida appear light pink when gram stained, indicating that this species is a gram-negative bacteria. Gram staining categorizes bacteria based on their cell wall constituents. Because the Pseudomonas putida are gram-negative, this means that they have very thin peptidoglycan cell walls. The lesser amount of peptidoglycan is not able to hold on to the crystal violet during the decoloring process, which gives gram-negative bacteria their characteristic pink color.
The cells are bacillus in morphology. This means they are rod-shaped, which we can clearly see in the images above. The cells are relatively evenly dispersed, though some have a streptobacilli arrangement. An example photo can be seen above in the top photo (http://microbe-canvas.com/Bacteria.php?p=686).
differentiating
differentiating
See the images below to learn about the tests that can be performed to determine if your unknown microbe is P. putida.
First row (left to right): blood agar, MacConkey agar, DNase, anaerobic chamber
Second row (left to right): Gram stain, phenol red broth, catalase, oxidase
Third row (left to right): Sulfide, Indole, Motility
This species is well differentiated. Even many related strains of Pseudomonas do not grow completely the same as Pseudomonas putida. This being said, similar strains can have some related tests. For example, Pseudomonas aeruginosa are also catalase and oxidase positive. A few differences between the species are that P. aeruginosa can grow at 42 degrees Celsius, they can convert nitrate to nitrite and they can convert nitrite to gas (P. putida is negative for all of these things). Another useful test to help distinguish between Pseudomonadacae and Enterobacteriacae is the oxidase test.
Pseudomonas putida is able to grow on normal blood agar, however it is negative for beta hemolysis so it will not appear yellow-ish on the plate. Some bacteria produce exotoxins which cleave red blood cells. This is the result we are looking for.
http://www.antimicrobe.org/clinicmicro/pseudomonas%20aerug1.htm
P. putida can grow on MacConkey agar but it is lactose negative, so the colonies will not take on a pink color. MacConkey agar selects for gram negative bacteria as well as lactose production. (NOTE: presents similar to P. aeruginosa)
P. putida is DNAse negative which means that DNAse does not depolymerize the DNA in this microbe.
https://laboratoryinfo.com/deoxyribonuclease-dnase-test/
P. putida is an aerobe, and it is negative for facultative which means it is an obligate aerobe. This means it requires oxygen at all times to grow.
https://microbeonline.com/cultivation-of-aerobic-and-anaerobic-bacteria/
P. putida is gram negative which means that it appears pink with a gram stain. Because the Pseudomonas putida are gram-negative, this means that they have very thin peptidoglycan cell walls.
P. putida is negative for glucose fermentation (left) which means that the broth would appear red in a phenol red broth test since no carbohydrate is being fermented. It is positive for glucose oxidation, but this test does not help us with that classification.
P. putida are catalase positive (top) which means that they are able to breakdown hydrogen peroxide into oxygen and water. This enzyme is present in nearly all aerobic bacteria.
P. putida are oxidase positive. This test is useful to differentiate between Pseudomonadacae and Enterobacteriaceae. It also selects for those bacteria which use oxygen as the final electron acceptor in the transport chain.
https://orbitbiotech.com/oxidase-and-indole-test-indole-oxidase-biochemical-tryptophan/
P. putida is hydrogen sulfide negative. A negative H 2 S test is denoted by the absence of blackening.
P. putida is indole negative. A yellow color denotes a negative indole test after addition of Kovacs Reagent.
https://orbitbiotech.com/oxidase-and-indole-test-indole-oxidase-biochemical-tryptophan/
P. putida are mobile A positive motility test is indicated by a diffuse zone of growth flaring from the line of inoculation.
https://microbeonline.com/tests-bacterial-motility-procedure-results/
How to grow
How to grow
This microbe was first documented by Gibson, DT in 1968 in his paper "Oxidative degradation of aromatic hydrocarbons by microorganisms. I. Enzymatic formation of catechol from benzene".
This is an aerobic microbe that can be grown on nutrient agar (23 g agar/1000ml DI water) at 30 degrees Celsius.
This microbe is characterized as a biosafety level 1 organism since it prevents minimal potential hazard to laboratorians and the environment.
molecular diagnostics
molecular diagnostics
Using 16S primers to PCR amplify and find the full 16S sequence:
Primers used:
Forward: PpF: 5’-CCA AAA CTG GCA AGC TAG AGT AC-3’;
Reverse: PpR: 5’-CAT CTC TGG AAA GTT CTC TGC-3’
Melting temperature:
Forward: 55.3°C
Reverse: 52.4°C
GC content:
Forward: (11/23)= 47.8%;
Reverse: (10/18) = 48%
We want primers to have between 40-60% GC content, so this primer set is good in terms of this factor.
After performing in silico PCR, the resulting partial 16S sequence was put into NCBI BLAST in order to acquire the full 16S sequence.
Full 16S FASTA sequence:
>NR_113651.1 Pseudomonas putida strain NBRC 14164 16S ribosomal RNA, partial sequence
ATTGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAGCGGATGAGAAGAGCTTGCTCTTCGATTCAGCGGCGGACGGGTGAGTAATGCCTAGGAATCTGCCTGGTAGTGGGGGACAACGTTTCGAAAGGAACGCTAATACCGCATACGTCCTACGGGAGAAAGCAGGGGACCTTCGGGCCTTGCGCTATCAGATGAGCCTAGGTCGGATTAGCTAGTTGGTGAGGTAATGGCTCACCAAGGCGACGATCCGTAACTGGTCTGAGAGGATGATCAGTCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGAAAGCCTGATCCAGCCATGCCGCGTGTGTGAAGAAGGTCTTCGGATTGTAAAGCACTTTAAGTTGGGAGGAAGGGCATTAACCTAATACGTTAGTGTTTTGACGTTACCGACAGAATAAGCACCGGCTAACTCTGTGCCAGCAGCCGCGGTAATACAGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCGCGTAGGTGGTTTGTTAAGTTGGATGTGAAAGCCCCGGGCTCAACCTGGGAACTGCATCCAAAACTGGCAAGCTAGAGTACGGTAGAGGGTGGTGGAATTTCCTGTGTAGCGGTGAAATGCGTAGATATAGGAAGGAACACCAGTGGCGAAGGCGACCACCTGGACTGATACTGACACTGAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCAACTAGCCGTTGGAATCCTTGAGATTTTAGTGGCGCAGCTAACGCATTAAGTTGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGCCTTGACATGCAGAGAACTTTCCAGAGATGGATTGGTGCCTTCGGGAACTCTGACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGTAACGAGCGCAACCCTTGTCCTTAGTTACCAGCACGTNATGGTGGGCACTCTAAGGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGGCCTGGGCTACACACGTGCTACAATGGTCGGTACAGAGGGTTGCCAAGCCGCGAGGTGGAGCTAATCTCACAAAACCGATCGTAGTCCGGATCGCAGTCTGCAACTCGACTGCGTGAAGTCGGAATCGCTAGTAATCGCGAATCAGAATGTCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCACCAGAAGTAGCTAGTCTAACCTTCGGGAGGACGGTTACCACGGTGTGATTCATGACTGGGGTGAAG
references
references
Fernández M, Porcel M, de la Torre J, Molina-Henares MA, Daddaoua A, Llamas MA, Roca A, Carriel V, Garzón I, Ramos JL, Alaminos M, Duque E. 2015. Analysis of the pathogenic potential of nosocomial Pseudomonas putida strains. Front Microbiol 6.
Kim SE, Park S-H, Park HB, Park K-H, Kim S-H, Jung S-I, Shin J-H, Jang H-C, Kang SJ. 2012. Nosocomial Pseudomonas putida Bacteremia: High Rates of Carbapenem Resistance and Mortality. Chonnam Med J 48:91–95.
Iwashita Y, Enokiya T, Suzuki K, Yokoyama K, Yamamoto A, Ishikura K, Okuda M, Imai H. 2013. Arbekacin treatment of a patient infected with a Pseudomonas putida producing a metallo-beta-lactamase. J Intensive Care 1.
Ruiz-Manzano A, Yuste L, Rojo F. 2005. Levels and Activity of the Pseudomonas putida Global Regulatory Protein Crc Vary According to Growth Conditions. J Bacteriol 187:3678–3686.
Gibson DT, Koch J R, Kallio R E, et al. 1968. Oxidative degradation of aromatic hydrocarbons by microorganisms. I. Enzymatic formation of catechol from benzene, p 2653-2662. In Biochemistry, 7th ed. doi: 10.1021/bi00847a031. PMID: 4298226.
Nakazawa T. 2002. Travels of a Pseudomonas, from Japan around the world. Environmental Microbiology 4:782–786.
Pseudomonas putida (Trevisan) Migula ATCC ® 700007TM. https://www.atcc.org/products/all/700007.aspx#generalinformation
CDC LC Quick Learn: Recognize the four Biosafety Levels. https://www.cdc.gov/training/quicklearns/biosafety/#:~:text=If%20you%20work%20in%20a,a%20nonpathogenic%20strain%20of%20E.
Altinok I. 2011. Multiplex PCR assay for detection of four major bacterial pathogens causing rainbow trout disease. Dis Aquat Org 93:199–206.
Scarpellini M, Franzetti L, Galli A. 2004. Development of PCR assay to identify Pseudomonas fluorescens and its biotype. FEMS Microbiology Letters 236:257–260.
about the researcher
about the researcher
My name is Katrina Russell and I am a third-year microbiology major and philosophy minor on the pre-med track at the University of Florida. I have aspirations to be a physician-scientist in the future and look forward to combining my knowledge of microbiology concepts and research methods to my passion for medicine. I chose to research into the microbe Pseudomonas putida because this is a bacteria I work with in my research lab. My goal was to gain a more complete understanding of the microbe and become more familiar with it.
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