The New York State Health Department (NYSDOH) is responsible for protecting and promoting the health of New Yorkers. The NYSDOH operates various programs and services that address public health issues such as infectious diseases, environmental health, chronic diseases, emergency preparedness, health care quality and access, and health equity. The NYSDOH also conducts research and surveillance to monitor and evaluate the health status and needs of the population.

One of the functions of the NYSDOH is to provide laboratory testing and identify pathogens that cause diseases in humans and animals. The NYSDOH operates the Wadsworth Center, the states public health laboratory and one of the nations most advanced biomedical research institutions. The Wadsworth Center performs various tests, from routine clinical diagnostics to specialized molecular and genomic analyses, to support diagnosing, preventing, and controlling infectious diseases.

One of the pathogens the Wadsworth Center tests for is Francisella tularensis subsp. tularensis, the causative agent of tularemia, is also known as rabbit or deer fly fever. Tularemia is a rare but potentially fatal zoonotic disease that can affect humans and animals. The NYSDOH monitors the occurrence and distribution of tularemia in New York State and provides guidance and resources for healthcare providers and the public on preventing and treating this disease.

This article will discuss some of the biochemical characteristics of Francisella tularensis subsp. tularensis is used to identify and differentiate from other subspecies and related bacteria. We will also review some of the methods and challenges of performing these tests in a laboratory setting.

Francisella tularensis is a gram-negative, facultative intracellular bacterium that causes tularemia, a zoonotic disease that affects humans and animals. Tularemia is also known as rabbit fever or deer fly fever, and various vectors, such as ticks, mosquitoes, flies, or contact with infected animals or contaminated water can transmit it.

There are four subspecies of Francisella tularensis, each with different geographic distributions and virulence levels. The most virulent and dangerous subspecies is Francisella tularensis subsp. tularensis, also known as type A or biovar A. This subspecies is endemic to North America and can cause severe and potentially fatal infections in humans and animals.

Francisella tularensis subsp. tularensis is a small, non-motile, non-spore-forming coccobacillus that measures about 0.2-0.7 micrometers in length and 0.2-0.4 micrometers in width. It has a thin lipopolysaccharide (LPS) layer that lacks the O antigen, which makes it resistant to complement-mediated killing and phagocytosis. Its capsule-like structure also helps it evade the host immune system and survive inside macrophages.

Francisella tularensis subsp. tularensis is an aerobic bacterium that can grow on various media, such as cysteine heart agar, chocolate agar, or buffered charcoal yeast extract (BCYE) agar. It requires cysteine or cystine as an essential amino acid for growth and produces acid from glucose. It is oxidase-negative, catalase-positive, indole-negative, urease-negative, and nitrate-reductase-negative. It can also hydrolyze casein and gelatin but not starch or esculin.

Francisella tularensis subsp. tularensis is highly infectious and can cause disease in as few as 10-50 organisms. The incubation period ranges from 1 to 14 days, depending on the exposure route and the bacteria dose. The clinical manifestations of tularemia vary depending on the site of infection, but they typically include fever, headache, malaise, lymphadenopathy, ulceroglandular lesions, pneumonia, septicemia, or meningitis. The mortality rate of untreated type A tularemia can reach up to 60%, but it can be reduced to less than 2% with appropriate antibiotic therapy.

Francisella tularensis subsp. tularensis is a Gram-negative coccobacillus, an aerobic bacterium that causes a severe tularemia disease. It is non-spore-forming, non-motile, and requires cysteine for growth. It is also a facultative intracellular bacterium, meaning it can survive and multiply within host cells, such as macrophages.

Some of the basic properties of Francisella tularensis subsp. tularensis are summarized in the table below:

| Property | Result |

| --- | --- |

| Capsule | Positive |

| Catalase | Positive |

| Flagella | Negative |

| Gas | Negative |

| Gelatin hydrolysis | Negative |

| Gram staining | Faintly stained Gram-negative |

| H2S (in cysteine-supplemented medium) | Positive |

| Indole | Negative |

| Motility | Negative |

| Nitrate reduction | Negative |

| NH3 produced in liquid media | Negative |

| Oxidase | Negative |

| Shape | Short, rod-shaped, or coccoid |

| Spore | Negative |

| Sodium ricinoleate solubility | Positive |

| Urease | Negative |

Francisella tularensis subsp. tularensis can be differentiated from other subspecies and subtypes of Francisella tularensis using molecular methods, such as PCR, sequencing, or genotyping . Some genetic markers can be used to identify Francisella tularensis subsp. tularensis are the 16S rRNA gene, the ISFtu2 insertion sequence, the RD1 deletion region, and the SNP A154T in the form gene.

Francisella tularensis subsp. tularensis is highly virulent and can cause fatal infections in humans and animals with very low doses of bacteria. It is endemic to North America and has been classified as a potential agent of biological terrorism. Therefore, it is important to understand its properties and characteristics to diagnose, treat, and prevent tularemia.

Fermentation of Francisella tularensis subsp. tularensis

Fermentation is a metabolic process that converts organic compounds into simpler molecules, such as acids, gases, or alcohols. Fermentation can identify bacteria based on their ability to ferment different substrates, such as sugars or amino acids.

Francisella tularensis subsp. tularensis is a non-fermentative bacterium, meaning it does not produce acid or gas from carbohydrates. This is one characteristic that distinguishes it from other subspecies of Francisella tularensis, such as subs. Holarctic and subs. novicida, which can ferment glucose and mannitol, respectively.

The non-fermentative nature of Francisella tularensis subsp. tularensis can be demonstrated by using various biochemical tests, such as:

• Phenol red broth test: This test uses a broth medium that contains carbohydrates (such as glucose, lactose, or sucrose) and a pH indicator (phenol red). If the bacterium can ferment the carbohydrate, it will produce acid and lower the pH of the medium, turning it yellow. If the bacterium cannot ferment the carbohydrate, the medium will remain red or pink due to alkaline products. Francisella tularensis subsp. tularensis does not change the color of the phenol red broth, indicating that it does not ferment any of the carbohydrates tested.

• Methyl red test: This test uses a broth medium that contains glucose and a pH indicator (methyl red). If the bacterium can ferment glucose, it will produce acid and lower the pH of the medium below 4.4, turning it red. If the bacterium cannot ferment glucose, the medium will remain yellow or orange due to neutral or alkaline products. Francisella tularensis subsp. tularensis does not turn the methyl red broth red, indicating that it does not ferment glucose.

• Voges-Proskauer test: This test uses a broth medium that contains glucose and two reagents (alpha-naphthol and potassium hydroxide). If the bacterium can ferment glucose, it will produce a neutral or alkaline end product called acetoin, which reacts with the reagents to form a red complex. If the bacterium cannot ferment glucose, no color change will occur. Francisella tularensis subsp. tularensis does not produce a red color in the Voges-Proskauer test, indicating that it does not ferment glucose.

These tests show that Francisella tularensis subsp. tularensis is a non-fermentative bacterium that does not utilize carbohydrates as an energy source. This is consistent with its intracellular lifestyle and preference for amino acids as carbon sources.

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Francisella tularensis subsp. tularensis is a gram-negative, non-motile, non-spore-forming, facultative intracellular bacterium that causes tularemia, a zoonotic disease that can affect humans and animals. One of the ways to identify and differentiate this subspecies from other Francisella subspecies is by performing biochemical tests that detect the presence or absence of certain enzymes.

Some of the enzymatic reactions of Francisella tularensis subsp. tularensis are:

• Catalase test: This test detects the presence of catalase, an enzyme that breaks down hydrogen peroxide into water and oxygen. Francisella tularensis subsp. tularensis is catalase-positive, producing bubbles when exposed to hydrogen peroxide.

• Oxidase test: This test detects the presence of cytochrome c oxidase, an enzyme that transfers electrons from a donor to oxygen, reducing it to water. Francisella tularensis subsp. tularensis is oxidase-negative, meaning it does not change the color of a paper strip impregnated with a reagent when exposed to oxygen.

• Urease test: This test detects the presence of urease, an enzyme that hydrolyzes urea into ammonia and carbon dioxide. Francisella tularensis subsp. tularensis is urease-negative, meaning it does not change the color of a broth or agar medium containing urea and a pH indicator when incubated.

• Indole test: This test detects the presence of tryptophanase, an enzyme that degrades tryptophan into indole, pyruvate, and ammonia. Francisella tularensis subsp. tularensis is indole-negative, meaning it does not produce a pink or red color when reacted with Kovac`s reagent after incubation in a tryptone broth.

• Nitrate reduction test: This test detects the ability of bacteria to reduce nitrate to nitrite or other nitrogenous compounds. Francisella tularensis subsp. tularensis is nitrate reduction-positive, producing a red color when treated with sulfanilic acid and dimethyl-alpha-naphthylamine after incubation in a nitrate broth.

These enzymatic reactions can help distinguish Francisella tularensis subsp. tularensis from other Francisella subspecies, such as Francisella tularensis subsp. Holarctic is catalase-negative, oxidase-positive, urease-positive, indole-positive, and nitrate reduction-negative.

However, biochemical tests alone are insufficient to confirm the identification of Francisella tularensis subsp. tularensis may vary depending on the strain, culture conditions, and reagents used. Therefore, other methods such as molecular techniques, serological tests, and animal inoculation are also recommended to diagnose and type this bacterium accurately.

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