3.3 Gram-Negative Bacilli & Enterobacteriaceae

Gram-Negative Bacilli & Enterobacteriaceae

Gram-negative bacilli (GNB) represent a massive and highly diverse group of organisms that stain pink or red on the Gram stain. They are responsible for a vast majority of clinical infections, including community-acquired urinary tract infections (UTIs), severe hospital-acquired pneumonias, intra-abdominal abscesses, and fatal bacteremias. The most clinically significant grouping within the GNB is the family Enterobacteriaceae (often simply referred to as enterics), though several clinically crucial "non-fermenters" also exist.

Defining Characteristics of Enterobacteriaceae

All members of the Enterobacteriaceae family share four fundamental defining characteristics that separate them from other Gram-negative rods:

1. Gram-Negative Bacilli: They possess an outer lipid membrane and a thin peptidoglycan layer.
2. Glucose Fermentation: They all universally possess the metabolic pathways to ferment glucose.
3. Oxidase Negative: They lack the cytochrome c oxidase enzyme (with the single exception of Plesiomonas shigelloides). This immediately separates them from Pseudomonas, Neisseria, and Campylobacter.
4. Nitrate Reduction: They possess the enzyme nitrate reductase, enabling them to reduce nitrates to nitrites.

Primary Differentiation: MacConkey Agar

When a Gram-negative rod is suspected, it is routinely and universally plated on MacConkey (MAC) agar. MAC is both selective (inhibiting Gram-positives via bile salts and crystal violet) and differential (detecting lactose fermentation via the neutral red pH indicator).

• Lactose Fermenters (LF): Rapidly ferment lactose, producing massive amounts of acid that drops the pH of the neutral red indicator, resulting in bright pink or red colonies. Key LF genera can be remembered by the mnemonic CEK: Citrobacter, Enterobacter, Klebsiella, and E. coli.
• Non-Lactose Fermenters (NLF): Cannot ferment lactose, so the pH does not drop, resulting in colorless or transparent colonies. Key NLF pathogens include Salmonella, Shigella, Proteus, Providencia, Morganella, and the non-fermenter Pseudomonas.

Triple Sugar Iron (TSI) Agar: The Workhorse Tube

TSI agar is a crucial, multi-parameter biochemical tool. The agar is poured into a tube on a slant with a deep butt. It contains three carbohydrates (glucose 0.1%, lactose 1%, sucrose 1%), phenol red (as a pH indicator; red = alkaline/K, yellow = acid/A), and sodium thiosulfate with ferrous sulfate (to detect H2S production).

Interpreting TSI Reactions:

• Alkaline slant / Acid butt (K/A): Red slant, yellow butt. Indicates the organism ferments glucose only. The small amount of glucose (0.1%) is quickly exhausted, and the organism shifts to metabolizing peptones on the aerobic slant, releasing alkaline amines that turn the slant back to red, while the anaerobic butt remains acidic. (Example: Shigella).
• Acid slant / Acid butt (A/A): Yellow slant, yellow butt. Indicates glucose fermentation AND fermentation of lactose and/or sucrose. The massive acid production from the 1% sugars keeps the entire tube yellow. (Example: E. coli, Klebsiella).
• Alkaline slant / Alkaline butt (K/K): Red slant, red butt. No carbohydrates are fermented. The organism utilizes only peptones. This indicates a strict non-fermenter, meaning the organism is not a member of Enterobacteriaceae. (Example: Pseudomonas aeruginosa).
• H2S Production (Blackening): The organism produces hydrogen sulfide gas, which reacts with iron to form a black precipitate (ferrous sulfide). This blackening always masks an acid (yellow) butt. Therefore, a tube that is K/Black is recorded as K/A, H2S+. (Example: Salmonella, Proteus).
• Gas Production: Bubbles, cracks, or complete displacement of the agar from the bottom of the tube indicate gas production during fermentation.

The IMViC Panel and Essential Biochemical Tests

The IMViC series is the traditional suite of foundational biochemical tests used to establish the identity of enteric organisms.

1. Indole: Determines the ability of the organism to produce the enzyme tryptophanase, which cleaves the amino acid tryptophan into indole. A positive test is indicated by a bright red ring forming at the surface of the broth after the addition of Kovac's or Ehrlich's reagent. E. coli is the classic strongly indole-positive organism.
2. Methyl Red (MR): Tests for the mixed acid fermentation pathway of glucose metabolism, which produces large volumes of stable acids. A positive result is a red color after adding the methyl red indicator, signifying a sustained pH drop below 4.4.
3. Voges-Proskauer (VP): Tests for the alternative butylene glycol pathway, which produces neutral end products like acetoin (acetylmethylcarbinol). A positive result is a pink/red color after adding alpha-naphthol and KOH. Generally, organisms are either MR positive OR VP positive; they are rarely both.
4. Citrate: Determines if the organism possesses transport enzymes to utilize citrate as its sole exogenous carbon source. Grown on Simmons citrate agar, a positive test is indicated by visible growth and a color change of the bromothymol blue indicator from green to vivid blue due to alkaline byproducts. (Examples: Klebsiella, Enterobacter).

Other Highly Important Tests:

• Urease: Detects the enzyme urease, which hydrolyzes urea into ammonia and CO2. The resulting ammonia dramatically raises the pH, turning the phenol red indicator an intense, brilliant pink. Proteus species are rapid and powerful urease producers.
• Motility: Evaluated in semi-solid agar or via wet mount. Most enterics are motile via peritrichous flagella, with two major exceptions: Klebsiella and Shigella are universally non-motile (Mnemonic: "SKY" - Shigella, Klebsiella, Yersinia (at 37C) are non-motile).
• Decarboxylases (Lysine, Ornithine, Arginine): Tests the ability to enzymatically remove the carboxyl group from specific amino acids in a Moeller base medium, leading to an alkaline pH shift (purple).
• Phenylalanine Deaminase (PAD): Detects the deamination of phenylalanine to phenylpyruvic acid. Addition of ferric chloride yields a dark green color. Positive for Proteus, Providencia, and Morganella.

Key Enterobacteriaceae Pathogens

Non-Fermenting Gram-Negative Bacilli

These organisms are strict aerobes that absolutely cannot ferment glucose (they oxidize it or are asaccharolytic) and are not part of the Enterobacteriaceae. They typically show a K/K reaction on TSI.

Pseudomonas aeruginosa

The most frequently isolated and clinically important non-fermenter. It is an opportunistic, nosocomial pathogen known for causing severe, life-threatening infections in cystic fibrosis patients, burn victims, and ventilator patients.

• Key Identifiers: Strict aerobe, fiercely oxidase positive, produces a characteristic pungent grape-like, corn tortilla, or sweet odor. It frequently produces pyocyanin (a water-soluble blue-green pigment) and pyoverdine (a fluorescent yellow-green pigment). It grows easily on MAC as a non-lactose fermenter and demonstrates significant intrinsic and acquired resistance to many classes of antimicrobial agents. It grows easily at 42°C, which helps distinguish it from other Pseudomonas species.