Laboratory Diagnosis and Biochemical Identification of Enterobacteriaceae

Microscopic Examination and Staining Characteristics

The microscopic diagnosis of Enterobacteriaceae begins with the Gram stain, which reveals gram-negative bacilli. These organisms typically appear as coccobacilli or straight rods with rounded ends. Specialized staining techniques are employed for specific genera. For instance, Yersinia species demonstrate a characteristic bipolar appearance, often described as a "closed safety pin," when treated with Wayson stain or Methylene Blue stain.

In cases of infections involving Klebsiella granulomatis, microscopic examination of lesions using Wright or Giemsa stains reveals the presence of Donovan bodies. These are characterized as blue rods with prominent polar granules, which also give rise to a safety-pin appearance. These bodies are typically surrounded by a large, distinctive pink capsule.

Primary and Selective Culture Media

Standard isolation usually involves the use of $5\,$ sheep blood agar, chocolate agar, and MacConkey agar. To isolate specific enteric pathogens, laboratories utilize more selective media. Hektoen enteric (HE) agar, xylose-lysine-deoxycholate (XLD) agar, and Salmonella-Shigella (SS) agar are commonly used. For the enrichment of Shigella and other Enterobacteriaceae, Gram-negative broth (GN) or Selenite broth (SEL) is employed.

Highly selective media for the isolation of Salmonella species include CHROMagar Salmonella, brilliant green agar, and bismuth sulfite agar. Bismuth sulfite agar is particularly useful for detecting the production of hydrogen sulfide ($H_2S$) through the use of ferrous sulfate; positive reactions for Salmonella species appear as a brown to black precipitate. On brilliant green agar, Salmonella species appear as white to pink or red colonies surrounded by a bright halo.

For the isolation of Yersinia enterocolitica from gastrointestinal specimens, cefsulodin-irgasan-novobiocin (CIN) agar is utilized. On this medium, Y. enterocolitica forms distinctive ‘bull’s-eye’ colonies after $48\,\text{hours}$ of incubation at room temperature. MacConkey-sorbitol agar (MAC-SOR) is a specialized tool used to differentiate sorbitol-negative Escherichia coli O157:H7 from other E. coli strains that can ferment this sugar alcohol.

General Colonial Morphology and Growth Patterns

Yersinia pestis exhibits unique growth characteristics. On $5\,$ sheep blood agar, colonies are pinpoint at $24\,\text{hours}$ but develop a rough, ‘cauliflower’ appearance by $48\,\text{hours}$. In broth cultures, Y. pestis displays a "stalactite pattern" where clumps of cells adhere to one side of the tube.

Colonial appearances on various media are diagnostic for different genera. On Eosin/methylene blue (EMB) agar, Escherichia coli is a lactose fermenter that forms blue-black colonies with a distinct metallic green sheen. Other coliform fermenters produce pink colonies, while nonfermenters appear translucent, amber, or colorless. Proteus species are known for their ability to ‘swarm’ depending on the agar concentration and produce a characteristic foul smell. Serratia marcescens may produce a red pigment, particularly when the culture plate is left at $25\,^{\circ}\text{C}$.

On Salmonella-Shigella (SS) agar, Shigella species appear colorless, whereas Salmonella species are colorless with a black center due to $H_2S$ production. On Xylose-lysine-deoxycholate (XLD) agar, Salmonella species appear colorless to red with a black center, Shigella species appear colorless to red, and other Enterobacteriaceae typically appear yellow.

Triple Sugar Iron (TSI) Agar

The Triple Sugar Iron (TSI) agar test is a fundamental biochemical screen that determines an organism's ability to ferment glucose, lactose, and sucrose, as well as its ability to produce hydrogen sulfide ($H_2S$). The medium contains a ratio of $10$ parts lactose to $10$ parts sucrose to $1$ part glucose, along with peptone, phenol red (pH indicator), and ferrous sulfate ($H_2S$ indicator).

An alkaline slant/no change in the butt ($K/NC$) or alkaline slant/alkaline butt ($K/K$) indicates the organism does not utilize glucose, lactose, or sucrose. An alkaline slant/acid butt ($K/A$) indicates the fermentation of glucose only. An acid slant/acid butt ($A/A$) indicates the fermentation of glucose and either sucrose, lactose, or both. The production of gas ($CO_2$ or $H_2$) is visible as bubbles or cracks in the agar, and $H_2S$ production is indicated by a black precipitate in the butt, which requires an acidic environment to form.

Quality control for TSI includes E. coli ($A/A$), Salmonella typhi ($K/A$, $H_2S+$), and Pseudomonas aeruginosa ($K/NC$). In reporting, a circle around the "A" for the butt (␀) typically denotes gas production.

Lysine Iron Agar (LIA)

Lysine Iron Agar (LIA) is used to determine if an organism decarboxylates or deaminates lysine and produces $H_2S$. The medium contains lysine, peptones, glucose, ferric ammonium citrate, and sodium thiosulfate. When glucose is fermented, the butt becomes acidic (yellow). If the organism produces lysine decarboxylase, the resulting cadaverine neutralizes the acids, reverting the butt to an alkaline purple state ($K/K$).

If oxidative deamination of lysine occurs, the reaction with ferric ammonium citrate and a coenzyme (flavin mononucleotide) produces a burgundy (Bordeaux red) color on the slant ($R/A$). If deamination does not occur, the slant remains purple. Blackening of the butt indicates $H_2S$ production. Quality control organisms include Salmonella typhimurium ($K/K$, $H_2S+$), Shigella flexneri ($K/A$), and Proteus vulgaris ($R/A$).

Phenylalanine Deaminase (PAD) and Indole Tests

The Phenylalanine Deaminase (PAD) test determines if an organism can deaminate phenylalanine to phenylpyruvic acid. This is performed on an agar slant with $0.2\,$ phenylalanine. After incubation, a $10\,$ ferric chloride ($FeCl_3$) reagent is added; a green color indicates a positive result. This test is crucial for differentiating the Proteeae tribe (Proteus, Morganella, and Providencia), which are positive, from other Enterobacteriaceae.

Indole production is tested using Sulfide-Indole-Motility (SIM) medium or specialized broth. Indole is a degradation product of tryptophan, formed by the enzyme tryptophanase. In the SIM medium, a red color after adding Kovac’s reagent signifies indole presence. The Ehrlich indole test is more sensitive and uses xylene for extraction followed by para-dimethylaminobenzaldehyde (PDAB) in Ehrlich’s reagent to produce a red color. A rapid indole test using p-dimethylaminocinnamaldehyde results in a blue-green color within $2\,\text{minutes}$ for positive results.

Citrate Utilization and Urea Hydrolysis

The citrate utilization test determines if an organism can use sodium citrate as its sole carbon source and inorganic ammonium salts as its sole nitrogen source. Growth on the medium leads to the release of ammonia, raising the pH. The bromthymol blue indicator changes from green ($\text{pH} < 7.0$) to blue ($\text{pH} > 7.6$). Klebsiella pneumoniae serves as a positive control, while E. coli is negative. Christensen’s citrate medium is an alternative using phenol red, which turns from yellow to pink in alkaline conditions.

Urea hydrolysis is detected using Christensen’s method to identify the enzyme urease. The hydrolysis of urea produces ammonia and $CO_2$. The resulting alkalinity shifts the phenol red indicator from light orange ($\text{pH } 6.8$) to magenta ($\text{pH } 8.1$). Proteus vulgaris is a positive control, and E. coli is negative.

Methyl Red (MR) and Voges-Proskauer (VP) Tests

The Methyl Red (MR) test identifies bacteria perform mixed acid fermentation. After incubation in glucose broth, MR indicator is added; a red color is positive, while yellow indicates the pH is at least $6.0$ (negative). The Voges-Proskauer (VP) test (Barritt’s method) determines if acids formed during fermentation are metabolized to 2,3-butanediol via an acetoin intermediate. Reagents include $\alpha$-naphthol (catalyst) and $40\,$ potassium hydroxide ($KOH$) or sodium hydroxide ($NaOH$). Acetoin is oxidized to diacetyl, which reacts to form a red complex. E. coli is $MR+$ and $VP-$, while Enterobacter cloacae is $MR-$ and $VP+$.

Nitrate and Nitrite Reduction

The nitrate reduction test evaluates an organism's ability to reduce nitrate to nitrite and potentially further to nitrogen gas ($N_2$). The addition of N,N-dimethyl-$\alpha$-naphthylamine and sulfanilic acid results in a red color if nitrite is present, confirming the first stage of reduction.