Citrobacter amalonaticus

Introduction

Citrobacter amalonaticus is a bacteria that is known as a human pathogen. This microbe was discovered relatively recently and therefore much research is still being conducted. Microbes in the Citrobacter genus and Citrobacter amalonaticus specifically are known for causing urinary tract infections.

Taxonomy

Kingdom: Bacteria
- Phylum: Proteobacteria
  - Class: Gammaproteobacteria
    - Order: Enterobacterales
      - Family: Enterobacteriaceae
        Genus: Citrobacter
        Species: C. amalonaticus

grow citribacter amalonaticus

How do you grow it?

1. What is the BSL level for your microbe?

The biosafety level for my microbe, Citrobacter amalonaticus, is 1. A biosafety level of 1 means that this microbe is low risk and presents little danger to the environment, lab, and experimenters. However, standard lab practices such as wearing personal protective equipment as needed, hand washing, and more should be followed.

2. What type of agar do you need?

According to the American Type Culture Collection, the type of agar needed for growing Citrobacter amalonaticus is nutrient agar. Nutrient agar is a growth medium that cultivates bacteria and other microbes.

3. Does it need to be in aerobic/anaerobic conditions?

Citrobacter amalonaticus needs to be grown in aerobic conditions.

4. What is the procedure for growing it once it arrives?

Once Citrobacter amalonaticus arrives, it needs to be rehydrated with 0.5 mL of #3 broth. Then, the contents of this vial must be transferred to another test tube with 5-6 mL of #3 broth. Then, use drops from the primary broth tube to inoculate either an agar plate or agar slant. Finally, incubate at 37 °C for at least 24 hours following rehydration.

5. Special instructions?

The American Type Culture Collection does not include any special instructions for growing Citrobacter amalonaticus.

History

1. When was the first documentation of it? Who discovered it?

Citrobacter amalonaticus was discovered by Viola M. Young. This microbe was first documented and described in 1971 after being found in the feces of hospital patients. The overall genus Citrobacter itself was discovered in 1932 due to them being common pathogens in the urinary tracts of hospital patients. A lot is still being learned about the overall Citrobacter genus and my specific microbe.

VISUALIZE citrobacter amalonaticus

After searching the internet, I was not able to find any pictures of my bacteria on a microscopic level from a credible source. Instead, I am using pictures of bacteria from the same genus that share many of the same characteristics and is closely related to Citrobacter amalonaticus.

1. The image shown above is a gram stain of the microbe Citrobacter freundii. The microbes found in the Citrobacter genus are similar in terms of their characteristics and therefore we see the same trends in my microbe, Citrobacter amalonaticus, as we do in C. freundii which we see above. We can clearly tell that the microbe is slightly both pink and red, indicating that it is gram negative. Gram negative bacteria like those in the Citrobacter genus display characteristics like having an outer membrane and a thin peptidoglycan layer.

2. The morphology of the cells is bacillus meaning that the microbe appears like straight rods at the microscopic level. Additionally, microbes in the Citrobacter genus are either a single rod or in pairs so they can be named bacillus or diplobacilli.

Here is an image from a Scanning Electron Microscope of C. freundii. Scanning electron microscopes allow us to see the surfaces of objects and their morphological features. We can observe the rod-shaped morphology in the image.

Above is an image from a Transmission Electron Microscope of C. freundii. Transmission electron microscopes allow us to see inside of objects as light is transmitted completely through. As mentioned before, some of the Citrobacter bacteria appears as a single rod and some come in pairs. The stain utilized was uranyl acetate and lead citrate.

Above is an image from Confocal immunofluorescence microscopy of C. freundii. This type of microscopy is utilized to locate an antigen or protein of interest. A dye was utilized.

DIAGNOSTIC TESTS

There are multiple tests that can be used to identify my microbe and differentiate it from others. Citrobacter amalonaticus is gram negative, positive for growth on ordinary blood agar, positive for growth on MacConkey agar, Mannitol Salt Agar test positive, DNAse test negative, Catalase positive, Oxidase negative, Phenol Red Broth positive, Sulfide production positive, Indole production positive, Motility test positive and Facultative.

A blood agar plate is a plate that includes 5% sheep’s blood. This test looks for an organism’s ability to produce hemolysins and we can characterize them as β – hemolysis, α-hemolysis, γ – hemolysis. My microbe is positive for this test and depicts gamma hemolysis.

The MacConkey test is used to select for the growth of gram-negative bacteria such as my microbe, which is positive for this test. The test is also able to differentiate based on bile and lactose metabolism. My microbe would appear pink for this test as shown in this picture as it is lactose fermenting.

My microbe is positive for the Mannitol Salt Agar test. A positive result can be observed by the yellow colonies in the agar plate. The Mannitol Salt Agar test is primarily used to isolate and differentiate Staphylococcus aureus and other pathogens. The phenol red indicator is yellow at a pH below 6 which we see for my microbe indicating a positive result.

The DNAse plate uses differential media to detect the presence of deoxyribonuclease activity. My microbe is negative for this test indicated by the lack of a colorless zone around the organism and its colonies.

My microbe is facultative anaerobic. This means that it can basically grow in the presence of absence of oxygen. It will use aerobic respiration to produce ATP if oxygen is present and ferment if it is not.

My microbe is gram negative. This means that it shades pink or red after gram staining because of the presence of a thin peptidoglycan layer as opposed to a thick one. We can observe this in the microscope slide shown to the right. Gram staining allows us to properly observe and identify organisms.

My microbe is positive for the phenol red broth test. With glucose, this test is utilized to test for the presence of fermentation reactions. A positive test will cause a color change from red to yellow like for my microbe.

My microbe is negative for the sulfide production test, positive for indole production test, and positive for the motility test. A negative sulfide production test does not produce a black precipitate, a positive indole production test produces a red ring, and a positive motility test creates visible turbidity.

My microbe is positive for the catalase test which essentially tests for the presence of catalase, an enzyme. Catalase is an enzyme frequently found in aerobic bacteria and it breaks hydrogen peroxide into water and oxygen as seen in the picture.

My microbe is negative for the catalase test which tests for the presence of a cytochrome oxidase system. Oxidase is sometimes found in the Electron Transport Chain of bacteria. A positive test will show purple shading, but a negative test will be colorless like we see in the picture for my microbe.

Disease and treatments

1. My microbe, Citrobacter amalonaticus, is known as an opportunistic pathogen, meaning that it does not typically cause disease in a healthy cost but can do so when the hosts immunity or resistance is weakened. This microbe is most known for causing urinary tract infections, gastroenteritis, and meningitis (1). It does not provide any sort of benefit to the host as it is purely pathogenic. The best ways to prevent contraction of this disease are to just perform proper hand-washing techniques and isolate yourself from those with the disease. Infection control and aseptic technique in general is key.

2. Citrobacter amalonaticus and other Citrobacter species are commonly found in animal and human intestinal tracts, certain foods, water, and even soil. The sources of this microbe whenever there is an outbreak or problem are the hands of the hospital staff and the gastrointestinal tracts of staff and patients.

3. My microbe, Citrobacter amalonaticus is important mainly because urinary tract infections are known as the most common health care associated infection and Citrobacter diseases account for around 10% of all UTI’s. Therefore, Citrobacter amalonaticus is important in researching because finding a cure or more information can be key in preventing hospital infections and saving lives. As mentioned earlier, this microbe is also opportunistic making it dangerous and important to research. Something else which is interesting is that many Citrobacter species play a role in the nitrogen cycle reducing nitrate to nitrite in our environment.

4. There are several antibiotics that work against Citrobacter amalonaticus and the Citrobacter genus in general. Amoxycillin and ampicillin are two antibiotics that are commonly utilized to treat gram-negative urinary tract infections, however there is significant antibiotic resistance with these. Trimethoprim and amoxiclav are other antibiotics also prescribed commonly but resistance to these antibiotics is also on the rise (4). Other antibiotics are also prescribed to treat urinary tract infections caused by Citrobacter amalonaticus. There are no other treatments besides antibiotics and there is currently no vaccine.

About the researcher

Hello everyone, my name is Sneh Patel and I am a third year Microbiology major. I am originally from Tampa, FL and have lived there for most of my life. I enjoy watching and playing sports such as basketball and football with my friends and family. I also love watching movies and shows.

References

Garcia V, Abat C, Moal V, Rolain JM. 2016. Citrobacter amalonaticus human urinary tract infections, Marseille, France. New Microbes and New Infections 11:1–5.

Ranjan KP, Ranjan N. 2013. Citrobacter: An emerging health care associated urinary pathogen. Urology Annals. Wolters Kluwer -- Medknow Publications.

Liu S, Kielian T. 2011. MyD88 is pivotal for immune recognition of Citrobacter koseri and astrocyte activation during CNS infection. Journal of Neuroinflammation 8:35.

Zhang J, Li J, Wu C, Liang X. 2010. Citrobacter freundii on corrosion behavior of HSn701-A in reclaimed water2010 International Conference on E-Product E-Service and E-Entertainment, ICEEE2010.

Badger JL, Stins MF, Kim KS. 1999. Citrobacter freundii invades and replicates in human brain microvascular endothelial cells. Infection and Immunity 67:4208–4215.

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Garcia V, Abat C, Moal V, Rolain JM. 2016. Citrobacter amalonaticus human urinary tract infections, Marseille, France. New Microbes and New Infections 11:1–5.

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