Enterobacteriaceae Cultural Characteristics

Enterobacteriaceae is a large and diverse family of Gram-negative bacteria that includes over 30 genera and more than 100 species. The name Enterobacteriaceae comes from the fact that many members of this family are commonly found in the intestines of animals and humans, where they are usually harmless or beneficial symbionts. However, some enterobacteria can also cause infections in various parts of the body, such as the urinary tract, respiratory tract, bloodstream, and wounds. Some of the most well-known pathogens in this family are Salmonella, Escherichia coli, Klebsiella, Shigella, Enterobacter, and Citrobacter.

Enterobacteriaceae are rod-shaped bacteria that can be motile or nonmotile. They have peritrichous flagella or fimbriae that help them attach to surfaces and hosts. They are facultative anaerobes, meaning they can grow with or without oxygen. They ferment sugars to produce acids and gases, such as lactic acid, ethanol, carbon dioxide, and hydrogen. They also reduce nitrate to nitrite, which can be detected by a colorimetric test. They do not produce cytochrome c oxidase, an enzyme involved in the electron transport chain. They are catalase-positive, meaning they can break down hydrogen peroxide into water and oxygen.

Enterobacteriaceae can be identified and classified by various biochemical tests and molecular methods. One of the most commonly used tests is the MacConkey agar, which is a selective and differential medium that inhibits the growth of Gram-positive bacteria and distinguishes between lactose-fermenting and non-lactose-fermenting enterobacteria based on the color of the colonies. Other tests include the indole test, the methyl red test, the Voges-Proskauer test, the citrate test, the urease test, and the triple sugar iron test. Molecular methods include polymerase chain reaction (PCR), DNA sequencing, and multilocus sequence typing (MLST).

Enterobacteriaceae are important for human health and disease. They play a role in maintaining the normal flora of the gut and modulating the immune system. They also produce vitamins such as vitamin K and B12 that are essential for human nutrition. However, they can also cause serious infections when they invade other tissues or organs or when they acquire antibiotic resistance genes. Some of the diseases caused by enterobacteria include gastroenteritis, typhoid fever, urinary tract infections, septicemia, pneumonia, meningitis, and plague. Some enterobacteria can also produce toxins that damage the host cells or interfere with their functions. For example, Shiga toxin produced by Shigella dysenteriae and some strains of E. coli causes bloody diarrhea and hemolytic uremic syndrome.

Enterobacteriaceae are widely distributed in nature and can be found in soil, water, plants, animals, and humans. They can also survive in various environmental conditions and adapt to different hosts. They can exchange genetic material with other bacteria through horizontal gene transfer mechanisms such as plasmids, transposons, bacteriophages, and integrons. This allows them to acquire new traits such as virulence factors and antibiotic resistance genes. Some of the most concerning enterobacteria are those that are resistant to carbapenems, a group of antibiotics that are usually reserved for treating severe infections caused by multidrug-resistant bacteria. These bacteria are called carbapenem-resistant Enterobacterales (CRE) and pose a serious threat to public health.

In this article, we will discuss the cultural characteristics of some of the most common enterobacteria that cause infections in humans. Cultural characteristics refer to the appearance and behavior of bacteria when they are grown on different types of media under specific conditions. These characteristics can help us identify and differentiate between different species or strains of enterobacteria.

Citrobacter freundii is a gram-negative rod-shaped bacterium that belongs to the family Enterobacteriaceae. It is a facultative anaerobe that can grow in both aerobic and anaerobic conditions. It is motile with peritrichous flagella and can ferment lactose and other sugars. It is commonly found in soil, water, sewage, and the intestinal tracts of animals and humans. It can cause opportunistic infections such as urinary tract infections, septicemia, meningitis, and diarrhea.

Citrobacter freundii can be cultured on various types of media to observe its morphological and biochemical characteristics. Some of the commonly used media are:

• Nutrient Agar: This is a general-purpose medium that supports the growth of most bacteria. Citrobacter freundii produces smooth, convex, translucent, or opaque grey colored colonies with a shiny surface and entire margin on this medium. The colonies may be mucoid or rough occasionally.
• Blood Agar: This is a differential medium that contains sheep or horse blood and can show hemolytic reactions of bacteria. Citrobacter freundii produces circular, flat, entire red colored colonies on this medium. It does not cause hemolysis of the blood cells.
• MacConkey Agar: This is a selective and differential medium that contains bile salts and crystal violet to inhibit the growth of gram-positive bacteria and lactose and neutral red to differentiate lactose fermenters from non-fermenters. Citrobacter freundii produces pink colored colonies on this medium after 24 hours of incubation. The pale-colored colonies turn pink after further 24 hours of incubation. This indicates that Citrobacter freundii is a late or slow lactose fermenter.
• Violet Red Bile Agar (VRBA): This is another selective and differential medium that contains bile salts and crystal violet to inhibit the growth of gram-positive bacteria and lactose and neutral red to differentiate lactose fermenters from non-fermenters. Citrobacter freundii produces red to pink flat colonies on this medium. It may also produce a green metallic sheen over the colonies.
• Xylose Lysine Deoxycholate (XLD) Agar: This is a selective and differential medium that contains xylose, lysine, deoxycholate, sodium thiosulfate, ferric ammonium citrate, and phenol red to differentiate various enteric bacteria based on their ability to ferment xylose, decarboxylate lysine, produce hydrogen sulfide, and change the pH of the medium. Citrobacter freundii shows partial to complete inhibition on this medium. The colonies that grow are yellow to yellow-red in color.
• Salmonella Shigella (SS) Agar: This is a selective and differential medium that contains bile salts and brilliant green to inhibit the growth of most gram-positive and some gram-negative bacteria and lactose, sucrose, sodium thiosulfate, ferric citrate, and neutral red to differentiate various enteric bacteria based on their ability to ferment sugars, produce hydrogen sulfide, and change the pH of the medium. Citrobacter freundii produces colorless colonies with grey or black centers on this medium.
• Eosin Methylene Blue (EMB) Agar: This is a selective and differential medium that contains eosin Y and methylene blue to inhibit the growth of gram-positive bacteria and lactose and sucrose to differentiate lactose fermenters from non-fermenters. Citrobacter freundii produces brown colored colonies on this medium without any metallic sheen.

E. coli (Escherichia coli) is a gram-negative, rod-shaped, facultative anaerobic bacterium that is commonly found in the intestinal tract of humans and animals. It can grow on various types of media and has distinctive features depending on the medium used. Some of the common media and their cultural characteristics of E. coli are:

• Nutrient Agar Medium (NAM): On this general-purpose medium, E. coli forms large, thick, greyish white, moist, smooth, opaque or translucent colonies with a diameter of 1-3 mm .
• Blood Agar Medium (BAM): On this enriched medium that contains sheep or horse blood, E. coli forms greyish white colonies with a diameter of 1-3 mm. The colonies are non-hemolytic, meaning they do not cause any lysis of the red blood cells in the medium.
• MacConkey Agar Medium (MAC): On this selective and differential medium that contains bile salts and lactose, E. coli forms pink to red-colored circular colonies with a diameter of 2-3 mm . The colonies are lactose-fermenting, meaning they produce acid from lactose and lower the pH of the medium. The acid production also causes a color change in the pH indicator present in the medium.
• Violet Red Bile Agar Medium (VRBA): On this selective and differential medium that contains bile salts and lactose, E. coli forms red to pink flat colonies with a diameter of 1-2 mm . The colonies are lactose-fermenting and produce a green metallic sheen over the colonies. The colonies also show blue fluorescence around them under UV light.
• Xylose Lysine Deoxycholate Agar Medium (XLD): On this selective and differential medium that contains xylose, lysine, deoxycholate and phenol red, E. coli forms yellow to yellow-red colonies with a diameter of 2-3 mm . The colonies are xylose-fermenting and produce acid from xylose and lower the pH of the medium. The acid production also causes a color change in the pH indicator present in the medium.
• Salmonella Shigella Agar Medium (SS): On this selective and differential medium that contains bile salts, lactose, sodium thiosulfate and ferric citrate, E. coli forms slight growth with pink to red-colored small circular colonies with a diameter of 1-2 mm . The colonies are lactose-fermenting and produce acid from lactose and lower the pH of the medium. The colonies do not produce hydrogen sulfide gas from sodium thiosulfate.
• Eosin Methylene Blue Agar Medium (EMB): On this selective and differential medium that contains eosin and methylene blue dyes, E. coli forms blue-black bull’s eye colonies with a metallic green sheen with a diameter of 2-3 mm . The colonies are lactose-fermenting and produce acid from lactose and lower the pH of the medium. The acid production also causes a color change in the dyes present in the medium.

Klebsiella oxytoca is a Gram-negative, rod-shaped, facultatively anaerobic bacterium that belongs to the Enterobacteriaceae family. It is closely related to Klebsiella pneumoniae, but can be distinguished by its ability to produce indole and to utilize citrate and malonate as carbon sources.

Klebsiella oxytoca can grow on various culture media, such as nutrient agar, blood agar, MacConkey agar, violet red bile agar (VRBA), XLD agar, Salmonella Shigella (SS) agar, EMB agar, and bile esculin agar. The following table summarizes the cultural characteristics of Klebsiella oxytoca on these media:

Klebsiella pneumoniae is a Gram-negative, non-motile, encapsulated, rod-shaped bacterium that belongs to the family Enterobacteriaceae. It is a common cause of nosocomial infections, such as pneumonia, urinary tract infections, septicemia, and wound infections. It is also a major contributor to antimicrobial resistance, as many strains produce extended-spectrum β-lactamases (ESBLs) and/or carbapenemases that render them resistant to most β-lactam antibiotics.

Klebsiella pneumoniae can grow on various culture media and exhibits different colony characteristics depending on the medium and the strain. Here are some examples of the cultural characteristics of Klebsiella pneumoniae on commonly used media:

• Nutrient Agar Medium (NAM): On this general-purpose medium, Klebsiella pneumoniae forms circular, dome-shaped, mucoid, translucent or opaque greyish white colonies with a diameter of 2-3 mm .
• Blood Agar Medium: On this enriched medium that contains 5% sheep blood, Klebsiella pneumoniae forms circular, dome-shaped, mucoid, translucent or opaque greyish white colonies with a diameter of 2-3 mm . There is no hemolysis (breakdown of red blood cells) around the colonies .
• MacConkey Agar Medium: On this selective and differential medium that inhibits the growth of Gram-positive bacteria and distinguishes between lactose-fermenting and non-lactose-fermenting bacteria, Klebsiella pneumoniae forms circular, convex, mucoid, pink to red-colored opaque colonies with a diameter of 2-3 mm . The pink to red color indicates that Klebsiella pneumoniae ferments lactose and produces acid that lowers the pH of the medium.
• Violet Red Bile Agar (VRBA): On this selective and differential medium that inhibits the growth of Gram-positive bacteria and distinguishes between lactose-fermenting and non-lactose-fermenting bacteria based on bile salt precipitation, Klebsiella pneumoniae forms large, mucous, and golden- or dull-yellow colored colonies . The golden- or dull-yellow color indicates that Klebsiella pneumoniae ferments lactose and produces acid that lowers the pH of the medium.
• Xylose Lysine Deoxycholate (XLD) Agar: On this selective and differential medium that inhibits the growth of Gram-positive bacteria and distinguishes between lactose-fermenting and non-lactose-fermenting bacteria based on pH indicators and hydrogen sulfide production, Klebsiella pneumoniae forms yellow colonies surrounded by yellow zones with precipitation zones . The yellow color indicates that Klebsiella pneumoniae ferments xylose and/or lactose and produces acid that lowers the pH of the medium. The precipitation zones are due to bile salt precipitation caused by acid production. Klebsiella pneumoniae does not produce hydrogen sulfide gas that would result in blackening of the colonies or the medium.
• Salmonella Shigella (SS) Agar: On this selective and differential medium that inhibits the growth of Gram-positive bacteria and distinguishes between lactose-fermenting and non-lactose-fermenting bacteria based on pH indicators and hydrogen sulfide production, Klebsiella pneumoniae forms pink or red to cream-colored, mucoid, opaque colonies . The pink or red to cream color indicates that Klebsiella pneumoniae ferments lactose and produces acid that lowers the pH of the medium. Klebsiella pneumoniae does not produce hydrogen sulfide gas that would result in blackening of the colonies or the medium.
• Eosin Methylene Blue (EMB) Agar: On this selective and differential medium that inhibits the growth of Gram-positive bacteria and distinguishes between lactose-fermenting and non-lactose-fermenting bacteria based on pH indicators and metallic green sheen production, Klebsiella pneumoniae forms circular, dome-shaped, mucoid, pink to purple colored translucent or opaque colonies with a diameter of 2-3 mm . The pink to purple color indicates that Klebsiella pneumoniae ferments lactose and produces acid that lowers the pH of the medium. Klebsiella pneumoniae does not produce a metallic green sheen that is characteristic of some strains of Escherichia coli.
• Bile Esculin Agar: On this selective and differential medium that inhibits the growth of Gram-positive bacteria and distinguishes between bacteria that can hydrolyze esculin in the presence of bile salts, Klebsiella pneumoniae shows growth with blackening of the medium within 4 hours . The blackening of the medium indicates that Klebsiella pneumoniae hydrolyzes esculin to esculetin and glucose, and esculetin reacts with ferric citrate in the medium to form a black complex.

These are some of the cultural characteristics of Klebsiella pneumoniae on different media. However, it is important to note that there may be variations in colony morphology and color depending on the strain, incubation time, temperature, and other factors. Therefore, it is advisable to use multiple media and biochemical tests to confirm the identification of Klebsiella pneumoniae.

Morganella morganii is a species of Gram-negative, facultative anaerobic, and oxidase-negative bacteria that belongs to the Enterobacteriaceae family. It is commonly found in the intestinal tract of humans and animals as normal flora, but it can also cause opportunistic infections such as urinary tract infections, wound infections, and septicemia.

Morganella morganii can be identified by its cultural characteristics on different types of media. Here are some examples:

• Nutrient Agar: Circular, dome-shaped, mucoid, opaque cream-colored colonies.
• Blood Agar: Off-white or cream-colored, smooth, convex, pinpoint white centers, shiny opaque colonies.
• MacConkey Agar: Flat colorless colonies (non-lactose fermenting).
• Violet Red Bile Agar (VRBA): Circular opaque colorless colonies.
• XLD Agar: Yellow colonies with acid production that changes the pH of the medium.
• Salmonella Shigella (SS) Agar: Dark blue, purple, or violet colonies with clear or pink edges.
• EMB Agar: Flat colorless colonies (non-lactose fermenting).
• Bile Esculin Agar: Growth with no blackening of the medium.

Morganella morganii can be distinguished from other members of the Enterobacteriaceae by its ability to produce indole and hydrogen sulfide, its resistance to colistin and cephalothin, and its susceptibility to ampicillin and nitrofurantoin.

Proteus mirabilis is a Gram-negative, rod-shaped bacterium that exhibits swarming motility and urease activity. It is a common cause of urinary tract infections, especially in patients with catheters or abnormal urinary tracts.

Proteus mirabilis can grow on various types of media, but its swarming behavior can interfere with the isolation of single colonies or the identification of other bacteria in a mixed sample. Therefore, some media may contain additives such as boric acid or cetrimide to inhibit or control swarming.

The following are some of the cultural characteristics of Proteus mirabilis on different media:

• Nutrient Agar: Pale white colonies as swarming growth with successive waves to form a thin filmy layer of concentric circles.
• Blood Agar: Pale white colonies as swarming growth with successive waves to form a thin sheer layer of concentric circles; swarming can be controlled by adding 0.1% boric acid.
• MacConkey Agar: Flat colorless colonies (non-lactose fermenting).
• Violet Red Bile Agar (VRBA): Light-purple color colonies surrounded by red color swarming.
• XLD Agar: Yellow, surrounded by yellow zones, translucent, black center.
• Salmonella Shigella (SS) Agar: Slight growth; pink to red-colored small circular colonies.
• EMB Agar: Luxuriant growth; Grey colored colonies (non-lactose fermenting).
• Bile Esculin Agar: Growth with no blackening of the medium.

Proteus vulgaris is a rod-shaped, Gram-negative bacterium that belongs to the family Enterobacteriaceae. It is an opportunistic pathogen that can cause urinary tract infections, wound infections, and septicemia in humans and animals. It is also known for its swarming motility on solid media, which is mediated by peritrichous flagella and regulated by environmental factors.

Proteus vulgaris can grow on various culture media and has the following characteristics on some commonly used media:

• Nutrient Agar Medium (NAM): Proteus vulgaris produces irregular colonies with a lenticular shape and a "beaten copper" surface. The colonies are colorless and have a finely striated edge. The swarming activity of Proteus vulgaris appears as concentric rings of growth arising from a single colony or from the initial inoculum .
• MacConkey Agar Medium: Proteus vulgaris produces flat, colorless colonies that do not ferment lactose. The colonies are 2-3 mm in diameter and have a smooth surface .
• Blood Agar Medium: Proteus vulgaris produces pale white colonies with a dome-shaped elevation and a pinpoint white center. The colonies are 2-3 mm in diameter and have a glistening surface. Proteus vulgaris does not produce hemolysis on blood agar .
• EMB Agar Medium: Proteus vulgaris produces flat, colorless colonies that do not ferment lactose. The colonies are 2-3 mm in diameter and have a smooth surface .
• XLD Agar Medium: Proteus vulgaris produces yellow colonies with black centers, surrounded by yellow zones. The colonies are translucent and mucoid with precipitation zones. Proteus vulgaris ferments xylose and produces hydrogen sulfide on XLD agar .
• Salmonella Shigella (SS) Agar Medium: Proteus vulgaris produces dark blue, purple, or violet colonies with clear or pink edges. The colonies are opaque and mucoid. Proteus vulgaris does not ferment lactose but produces hydrogen sulfide on SS agar .
• Bile Esculin Agar Medium: Proteus vulgaris produces growth with no blackening of the medium. Proteus vulgaris does not hydrolyze esculin in the presence of bile salts .

Providencia stuartii is a gram-negative bacillus that is commonly found in soil, water, and sewage. It is the most common of the five species in the genus Providencia capable of causing human infections. Providencia stuartii is an opportunistic pathogen that can cause urinary tract infections, wound infections, septicemia, and diarrhea, especially in patients with severe burns, long-term indwelling catheters, or compromised immune systems.

Providencia stuartii can be cultured in nutrient agar or Columbia agar, with optimal growth at 37°C and a mesophilic temperature range. It is of risk group 2 on the biosafety level. Some other important characteristics of P. stuartii are:

• It is motile via flagella
• It is non-sporulating
• It is non-lactose fermenting
• It is catalase positive and oxidase negative
• It can grow in anaerobic conditions and on Simmon’s Citrate Agar

The following table summarizes the cultural characteristics of P. stuartii on different types of media:

Salmonella enterica is a gram-negative, rod-shaped, flagellated bacterium that can cause various diseases in humans and animals, such as enteric fever, gastroenteritis, septicemia, and typhoid fever. Salmonella enterica has more than 2600 serovars that differ in their antigenic structure and host specificity. Some of the most common serovars that cause human infections are Typhimurium, Enteritidis, Heidelberg, and Newport.

Salmonella enterica can be transmitted to humans through contaminated foods of animal origin, such as poultry, eggs, pork, and fish. Salmonella enterica can also survive in the environment for weeks or months under favorable conditions.

Salmonella enterica has the following cultural characteristics on different media:

• Nutrient Agar: Smooth colorless colonies with 2-4 mm diameter.
• Blood Agar: Non-hemolytic smooth white colonies.
• Mac-Conkey Agar: Transparent colorless colonies with no zone of precipitation; non-lactose fermenting colonies .
• Violet Red Bile Agar (VRBA): Colorless to orangish-yellow non-lactose fermenting colonies.
• XLD Agar: Red colonies with black centers; media itself turn red due to growth.
• Salmonella Shigella (SS) agar: Colorless colonies with black centers.
• EMB agar: Luxuriant growth; colorless non-fermenting colonies; some strains might be inhibited.

Salmonella enterica can be identified by its biochemical reactions, such as:

• Catalase: Positive
• Oxidase: Negative
• Indole: Negative
• Methyl Red: Positive
• Voges-Proskauer: Negative
• Citrate: Positive
• Urease: Negative
• Hydrogen Sulfide: Positive
• Lysine Decarboxylase: Positive
• Ornithine Decarboxylase: Positive

Salmonella enterica can also be serotyped by its somatic (O), flagellar (H), and capsular (Vi) antigens using specific antisera.

Serratia marcescens is a Gram-negative rod-shaped bacterium that belongs to the family Yersiniaceae. It is well known for the production of a bright red pigment called prodigiosin, which gives it a distinctive appearance on some culture media. However, not all strains of S. marcescens produce this pigment, and its production depends on several factors such as temperature, pH, and oxygen availability. S. marcescens is an opportunistic pathogen that can cause various infections in humans and animals, especially in hospital settings where it can contaminate medical devices and equipment.

The following table summarizes the cultural characteristics of S. marcescens on different types of media:

Shigella dysenteriae is a Gram-negative, non-motile, non-spore forming, rod-shaped bacterium that causes bacillary dysentery or shigellosis in humans. It belongs to subgroup A of the genus Shigella, which comprises four species: S. dysenteriae, S. flexneri, S. boydii, and S. sonnei. Shigella dysenteriae is the most virulent and invasive species of Shigella, and it produces a potent exotoxin called Shiga toxin that can cause severe complications such as hemolytic uremic syndrome.

Shigella dysenteriae can grow on various culture media, but it is usually isolated from stool samples using selective and differential media such as MacConkey agar, Xylose Lysine Deoxycholate (XLD) agar, Salmonella-Shigella (SS) agar, or Deoxycholate Citrate Agar (DCA) agar. The following table summarizes the cultural characteristics of Shigella dysenteriae on some commonly used media:

Shigella dysenteriae can also be identified by its biochemical and serological properties. Some of the biochemical tests that can be used to differentiate Shigella dysenteriae from other Enterobacteriaceae are:

• Indole test: Negative
• Methyl red test: Positive
• Voges-Proskauer test: Negative
• Citrate utilization test: Negative
• Urease test: Negative
• Lysine decarboxylase test: Negative
• Ornithine decarboxylase test: Negative
• Motility test: Negative

Shigella dysenteriae can be further classified into 15 serotypes based on the antigenic structure of its O (somatic) antigens. The most common serotype associated with outbreaks of shigellosis is serotype 1 (S. dysenteriae type 1), which is also the only serotype that produces Shiga toxin.

Yersinia pestis is the causative agent of plague, a highly fatal zoonotic disease that has caused three major pandemics in human history. Yersinia pestis is a gram-negative rod or coccobacillus that shows bipolar staining with Giemsa or Wright stains. It is a facultative anaerobe that can grow on ordinary media at 28°C (optimal) or 37°C (slow). It produces a capsule and several virulence factors, such as Yersinia murine toxin (ymt), plasminogen activator (pla), and type III secretion system (T3SS).

The cultural characteristics of Yersinia pestis on different media are as follows:

• Nutrient Agar Medium (NAM): Tiny, almost invisible, shiny grey, translucent “spots” after 24 hours; 1 to 2 mm irregular, grey-white to slightly yellow in color with raised, irregular, “fried egg” appearance, which becomes prominent as the culture ages .
• Blood Agar Medium (BAM): White or cream-colored, opaque colonies with entire margin; no hemolysis .
• MacConkey Agar Medium (MAC): Transparent colorless colonies with no zone of precipitation; non-lactose fermenting colonies .
• Violet Red Bile Agar (VRBA): Circular opaque colorless colonies .
• Xylose Lysine Deoxycholate Agar (XLD): Yellow colonies with acid production that changes the pH of the medium .
• Cetrimide Agar: Pink colonies, with or without purple centers.
• Eosin Methylene Blue Agar (EMB): Transparent colorless colonies with no zone of precipitation; non-lactose fermenting colonies .
• Bile Esculin Agar: Luxuriant growth with blackening of the medium .

Broth cultures of Yersinia pestis exhibit a characteristic “stalactite pattern” in which clumps of cells adhere to one side of the tube.

Yersinia pestis can be identified by biochemical tests such as catalase (positive), oxidase (negative), indole (negative), urease (negative), citrate utilization (negative), methyl red (positive), Voges-Proskauer (negative), and motility (negative). Serological tests such as agglutination and ELISA can also be used to detect specific antibodies or antigens. Molecular methods such as PCR and DNA sequencing can provide rapid and accurate identification and typing of Yersinia pestis strains.

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