Yersinia Selective Agar- Composition, Principle, Preparation, Results, Uses

Yersinia enterocolitica is a gram-negative, bacillus-shaped bacterium that belongs to the family Yersiniaceae. It is one of the three pathogenic species of the genus Yersinia, along with Y. pestis and Y. pseudotuberculosis.

Y. enterocolitica causes yersiniosis, a zoonotic disease that affects humans and various animals, such as pigs, cattle, deer, and birds. The infection can manifest as acute diarrhea, mesenteric adenitis, terminal ileitis, and pseudoappendicitis. In rare cases, it can also cause sepsis.

Y. enterocolitica is widely distributed in nature and can be found in various reservoirs, such as the intestinal tracts of mammals, avian species, cold-blooded species, and environmental water. However, not all strains of Y. enterocolitica are pathogenic for humans. The pathogenic strains are classified into six biogroups (1B and 2-5) and more than 57 serogroups based on their phenotypic and antigenic characteristics.

The most common serogroups associated with human disease are O:3 (biogroup 4), O:5,27 (biogroups 2 and 3), O:8 (biogroup 1B), and O:9 (biogroup 2). The serogroup distribution varies geographically; for example, serogroup O:3 is predominant in Europe, while serogroup O:8 is more prevalent in the United States.

The isolation and identification of Y. enterocolitica from clinical specimens and food samples can be challenging due to its low infectious dose, slow growth rate, and similarity to other enteric bacteria. Therefore, selective media and enrichment procedures are often used to enhance its recovery and differentiation.

Yersinia Selective Agar is a differential and selective medium that allows the isolation of Yersinia enterocolitica from other bacteria in a sample. The principle of this medium is based on the ability of Y. enterocolitica to ferment mannitol, a sugar that is present in the medium along with a pH indicator called neutral red. Mannitol fermentation produces acidic by-products that lower the pH of the medium around the colonies. Neutral red changes color from yellow to red in acidic conditions, making the mannitol-fermenting colonies appear red or pink on the agar. In addition, some mannitol-fermenting bacteria may also cause bile precipitation around their colonies, forming a clear zone in the otherwise turbid medium.

Yersinia Selective Agar also contains sodium deoxycholate and crystal violet, which are inhibitory agents that suppress the growth of gram-positive and some gram-negative bacteria. Furthermore, the medium is supplemented with cefsulodin and novobiocin, two antibiotics that selectively inhibit most Enterobacteriaceae except Yersinia spp. These ingredients make the medium highly selective for Y. enterocolitica and other Yersinia species.

Yersinia enterocolitica colonies on Yersinia Selective Agar are typically dark red with a translucent border and may be surrounded by a zone of precipitated bile. They are also called "bull`s eye" colonies because of their appearance. The colony size, smoothness, and ratio of the border to center diameter may vary among different serotypes of Y. enterocolitica. Other Yersinia species, such as Y. pseudotuberculosis, may also grow on this medium but they usually form smaller and paler colonies than Y. enterocolitica.

Other bacteria that do not ferment mannitol or are inhibited by the selective agents will either not grow or form colorless and translucent colonies on Yersinia Selective Agar. However, some bacteria, such as Serratia liquefaciens, Citrobacter freundii, and Enterobacter agglomerans, may resemble Y. enterocolitica on this medium and require further biochemical tests for confirmation.

Yersinia Selective Agar is a differential and selective medium that contains the following ingredients:

• Mannitol: This is a sugar that serves as a fermentable carbohydrate source for bacteria. Mannitol fermentation produces acid, which lowers the pH of the medium and causes a color change in the presence of neutral red indicator.
• Neutral Red: This is a pH indicator that turns red in acidic conditions and yellow in alkaline conditions. It helps to differentiate between mannitol fermenting and non-fermenting bacteria on the medium.
• Sodium Deoxycholate and Crystal Violet: These are bile salts and a dye that inhibit the growth of gram-positive and some gram-negative bacteria, making the medium selective for Yersinia and other enteric pathogens.
• Cefsulodin and Novobiocin: These are antibiotics that are added to the medium after sterilization to enhance its selectivity for Yersinia enterocolitica. Cefsulodin inhibits most gram-negative bacteria except for Yersinia, Pseudomonas, and Acinetobacter. Novobiocin inhibits most gram-positive bacteria and some gram-negative bacteria such as Proteus, Providencia, and Morganella.
• Sodium Chloride: This maintains the osmotic balance of the medium and provides essential ions for bacterial growth.
• Sodium Pyruvate and Magnesium Sulfate: These are growth factors that stimulate the growth of Yersinia enterocolitica and other bacteria on the medium.
• Agar: This is a polysaccharide derived from seaweed that solidifies the medium and provides a firm surface for bacterial growth and isolation.

The final pH of the medium is 7.4±0.2 at 25°C. The medium appears light pink in color before inoculation.

To prepare Yersinia Selective Agar, you will need the following materials:

• Yersinia Selective Agar powder (29.02 grams)
• Distilled water (500 ml)
• Yersinia Selective Supplement or Cefsulodin and Novobiocin solution (10 ml)
• Sterile Petri plates
• Autoclave
• Water bath
• Pipette
• Inoculating loop or swab
• Incubator

Follow these steps to prepare and use Yersinia Selective Agar:

1. Suspend 29.02 grams of Yersinia Selective Agar powder in 500 ml of distilled water in a suitable container. Mix well to dissolve the medium completely.
2. Sterilize the medium by autoclaving at 15 lbs pressure (121°C) for 15 minutes.
3. Cool the medium to 45-50°C in a water bath and aseptically add 10 ml of Yersinia Selective Supplement or Cefsulodin and Novobiocin solution. Mix well before pouring into sterile Petri plates.
4. Streak the specimen as soon as possible after it is received in the laboratory. The streak plate is used primarily to isolate pure cultures from specimens containing mixed flora.
5. Alternatively, if the material is being cultured directly from a swab, roll the swab over a small area of the surface at the edge; then streak from this inoculated area.
6. Incubate the plates at 22-32°C for 24-48 hours or suspend the sample (food, feces, etc.) in sterile Phosphate Buffer Saline and incubate for up to 21 days at 4°C.
7. Periodically subculture samples onto fresh Yersinia Selective Agar plates and incubate as above.
8. Examine the plates for typical colonies of Yersinia enterocolitica and other organisms.

The appearance of colonies on Yersinia Selective Agar depends on the ability of the bacteria to ferment mannitol and their susceptibility to the antibiotics cefsulodin and novobiocin. The medium also contains neutral red, which acts as a pH indicator and changes color from yellow to red in acidic conditions.

• Yersinia enterocolitica colonies appear translucent or translucent with dark pink centers. Colony edges are entire or irregular. After 48-hour incubation, colonies appear dark pink with a translucent border and may be surrounded by a zone of precipitated bile. This is because Y. enterocolitica is able to ferment mannitol and produce acid, which lowers the pH of the medium and causes the neutral red to turn red. The acid also precipitates the bile salts present in the medium, forming a clear zone around the colonies. Y. enterocolitica is also resistant to cefsulodin and novobiocin, which allows it to grow on the selective medium.
• Other Yersinia species, such as Y. pseudotuberculosis, Y. frederiksenii, Y. kristensenii, and Y. intermedia, may also grow on Yersinia Selective Agar, but they have different colony characteristics. These species are mannitol negative, meaning they do not ferment mannitol and do not produce acid. Therefore, their colonies are colorless and translucent, without any zone of precipitated bile. They may also have different shapes and sizes depending on the serotype. These species are also resistant to cefsulodin and novobiocin.
• Non-Yersinia organisms are usually inhibited by the selective agents in the medium, such as sodium deoxycholate, crystal violet, cefsulodin, and novobiocin. However, some bacteria may still grow on Yersinia Selective Agar, but they can be distinguished from Yersinia by their colony morphology and biochemical tests. For example, Serratia liquefaciens, Citrobacter freundii, and Enterobacter agglomerans may resemble Y. enterocolitica, but they can be identified by their ability to produce gas from glucose or lactose, which Yersinia does not. Other bacteria that may grow on Yersinia Selective Agar include Proteus spp., Klebsiella spp., Escherichia coli, and Salmonella spp., but they usually have different colony colors, shapes, and sizes.

To confirm the identity of suspicious isolates from Yersinia Selective Agar, further tests are necessary, such as biochemical tests (e.g., urease test, indole test, motility test), serological tests (e.g., slide agglutination test), and molecular tests (e.g., PCR). A less selective medium, such as MacConkey Agar, should also be inoculated with the specimen for the detection of other pathogens involved in the infection.

Yersinia Selective Agar is a useful medium for the selective isolation and enumeration of Yersinia enterocolitica from clinical specimens and food samples. Y. enterocolitica is an important foodborne pathogen that can cause gastroenteritis, mesenteric lymphadenitis, terminal ileitis, and septicemia in humans and animals. The pig is the major reservoir of pathogenic Y. enterocolitica of bioserotype 4/O:3, the most common type found in humans. Y. enterocolitica has been isolated from raw meats such as beef, lamb, pork, and chicken, but can also be found in cooked, pre-packaged deli meats. Consumption of raw or improperly cooked pork is the main source of gastroenteritis in humans, with the specific association with improperly prepared and handled pork chitterlings. Drinking unpasteurized milk or untreated water, or contact with infected animals also can be the source of infection.

Yersinia Selective Agar can also be used for the isolation of Yersinia species other than Y. enterocolitica, e.g., for Y. pseudotuberculosis. Y. pseudotuberculosis is another foodborne pathogen that can cause similar symptoms as Y. enterocolitica, but also can lead to reactive arthritis and erythema nodosum. Y. pseudotuberculosis has been isolated from various animals, such as rodents, birds, cats, dogs, and livestock, and from environmental sources, such as soil and water. The main route of transmission to humans is through ingestion of contaminated food or water.

Yersinia Selective Agar is a convenient and reliable medium for detecting pathogenic Yersinia species in natural samples. It has several advantages over other media, such as:

• It differentiates between mannitol fermenting and non-fermenting bacteria by using neutral red as a pH indicator.
• It inhibits gram-positive and a number of gram-negative bacteria by using sodium deoxycholate and crystal violet as selective agents.
• It enhances the growth of Yersinia species by using sodium pyruvate and magnesium sulfate as stimulants.
• It allows the identification of Y. enterocolitica by using cefsulodin and novobiocin as antibiotic supplements.

Yersinia Selective Agar is therefore a valuable tool for the diagnosis and surveillance of yersiniosis in humans and animals.

• Yersinia Selective Agar is a highly selective medium that inhibits the growth of most gram-positive and gram-negative bacteria, except for some species of Yersinia and a few other genera. However, this also means that it may miss other potential pathogens that may coexist with Yersinia enterocolitica in the specimen or sample. Therefore, it is advisable to use another less selective medium, such as MacConkey Agar, in parallel with Yersinia Selective Agar and incubate it at a higher temperature (35 +/- 2°C) to detect other possible causative agents of infection.
• Yersinia Selective Agar relies on the fermentation of mannitol as the main differential criterion for identifying Yersinia enterocolitica colonies. However, some strains of Yersinia enterocolitica may be weakly mannitol positive or negative, and some strains of other bacteria, such as Serratia liquefaciens, Citrobacter freundii, and Enterobacter agglomerans, may be mannitol positive and resemble Yersinia enterocolitica on the medium. Therefore, it is necessary to perform further biochemical and serological tests to confirm the identity of the isolates and rule out false positives or negatives.
• Yersinia Selective Agar may not be sufficient for the isolation of Yersinia enterocolitica from some specimens or samples that have a low number of viable cells or a high level of competing flora. In such cases, a "cold enrichment" procedure may be required to enhance the recovery of Yersinia enterocolitica. This involves suspending the specimen or sample in sterile phosphate buffer saline and incubating it at 4°C for up to 21 days before subculturing onto Yersinia Selective Agar. However, this method is time-consuming and may not be feasible in some situations.
• Yersinia Selective Agar may also support the growth of other species of Yersinia besides Yersinia enterocolitica, such as Yersinia pseudotuberculosis, Yersinia frederiksenii, Yersinia kristensenii, and Yersinia intermedia. These species may have different clinical significance and epidemiology than Yersinia enterocolitica and may require different treatment and prevention strategies. Therefore, it is important to differentiate them from Yersinia enterocolitica based on additional characteristics, such as motility, urease production, indole reaction, ornithine decarboxylase activity, and serotyping.
• Yersinia Selective Agar is not intended for use in the diagnosis of disease or other conditions in humans. It is only a screening tool for the isolation and enumeration of Yersinia enterocolitica from clinical specimens and food samples. The results obtained from this medium should be interpreted in conjunction with other clinical and laboratory findings and should not be used as the sole basis for diagnosis or treatment decisions.

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