Klebsiella pneumoniae: Lab Identification and Biochemical Tests

A patient with pneumonia coughs up thick sputum the color of red currant jelly. For over a century, that color has pointed toward one organism. But by the time the plate reaches your bench, the color is gone, and you see plain mucoid colonies. Several bacteria can look like this on the plate. Your job now is to prove the colony is Klebsiella pneumoniae and not another organism, using a short set of tests that each rule something out.

This page is about making that identification: which tests to run, what each result means, and the points where students read the results wrong.

Overview

Klebsiella pneumoniae is a Gram-negative rod of the family Enterobacteriaceae. It lives as normal flora in the human intestine and is also found widely in the environment. Most of the time it is harmless. The problem comes when it reaches a site it does not belong in, such as the lung, the urinary tract, or the blood, especially in a hospital patient.

It was first isolated in the late 19th century and was originally called Friedländer's bacillus. The classic disease linked to it is a severe pneumonia that produces thick, brick-red sputum described as "currant jelly." That presentation is now uncommon, but the name has stuck in textbooks and exams.

K. pneumoniae causes infection most often in infants, the elderly, people with weakened immunity, and those with heavy alcohol use. It is one of the leading causes of hospital-acquired infection. Its range includes pneumonia (it is a frequent cause of ventilator-associated pneumonia), urinary tract infection, bloodstream infection, and liver abscess. It causes only a small share of all pneumonia, but when it does cause pneumonia the illness can be severe.

The reason it matters so much today is resistance. Strains producing extended-spectrum beta-lactamases (ESBLs) or carbapenemases are among the global priority resistant pathogens.

What Klebsiella pneumoniae looks like, and what that tells you

Figure: mucoid colonies of K. pneumoniae

Klebsiella pneumoniae is a Gram-negative, non-spore-forming rod and a facultative anaerobe. Four features do most of the identification work, and each one also tells you what the organism is not.

It is oxidase-negative. This is the first gate. A Gram-negative rod that grows on MacConkey agar and is oxidase-negative is very likely an enteric organism (Enterobacteriaceae). If your isolate is oxidase-positive, it is not Klebsiella; think of Pseudomonas instead, and stop the enteric workup. This is why "is Klebsiella oxidase positive or negative" is the right question to ask early: a positive result changes everything.

It is catalase-positive. Like almost all Enterobacteriaceae. On its own this does not narrow things much, but a catalase-negative Gram-negative rod would argue against Klebsiella.

It ferments lactose, so it is pink on MacConkey. And the colonies are strikingly mucoid, wet, and glistening, because of a thick polysaccharide capsule. That mucoid look is the single most recognizable thing about Klebsiella on a plate. A pink, dry colony is more likely E. coli; a pink, mucoid, dome-shaped colony points to Klebsiella.

It is non-motile. This is unusual and useful. Most Enterobacteriaceae are motile; Klebsiella is not. It has no flagella, and therefore no H antigen. In a motility test it stays put. This one property separates Klebsiella from many look-alikes in the family, so "is Klebsiella motile" is a high-value question: the answer is no, and that no is diagnostic.

Hold these four together and you have a working profile before any tube test: oxidase-negative, catalase-positive, lactose-positive with mucoid colonies, non-motile.

Virulence factors and how Klebsiella causes disease

Klebsiella pneumoniae is usually harmless in the gut. It causes disease when it reaches a site it does not belong in, such as the lung, urinary tract, or blood, and once there, a set of virulence factors lets it survive and spread. Each factor below is tied to what it actually does in the body.

Figure: Virulence factors of Klebsiella pneumoniae

Capsule (the main one). The thick polysaccharide capsule is the single most important virulence factor. It coats the organism and blocks phagocytosis, so white blood cells cannot easily engulf it, and it protects against killing by complement. This is why Klebsiella infections can progress quickly in a weakened host. The capsule is also what makes the colonies mucoid on the plate, so the thing you see on the agar and the thing that makes the organism dangerous are the same feature. Certain capsule types (notably K1 and K2) are linked to the more aggressive, community-acquired infections such as liver abscess.

Hypervirulent strains and the string test. Some strains produce so much capsule that the growth becomes stringy. Touch a colony with a loop and lift, and it stretches into a long thread more than about 5 mm. A positive string test flags a hypervirulent strain, which is more likely to cause invasive disease such as liver abscess and to spread to distant sites (for example, the eye or the brain), even in a person who was previously healthy.

Lipopolysaccharide (endotoxin). The outer membrane LPS is the classic Gram-negative endotoxin. When the organism reaches the blood, LPS triggers a strong inflammatory response. In severe infection this drives the fever, low blood pressure, and organ dysfunction of sepsis and septic shock.

Fimbriae (adhesins). Surface fimbriae let the organism stick to host cells and to plastic surfaces. Type 1 fimbriae help it attach in the urinary tract, which is central to urinary infection. Type 3 fimbriae help it stick to medical devices such as catheters and ventilator tubing and build biofilm, which is why Klebsiella is such a common cause of device-associated hospital infection.

Siderophores. Iron is scarce inside the human body because the host locks it away. Klebsiella releases siderophores, molecules that scavenge iron from host proteins, so it can grow where iron is limited. Hypervirulent strains make extra siderophores, which helps them spread beyond the first site of infection.

Putting it together: the pathogenesis story

The sequence is the same across most Klebsiella infections. The organism first attaches using fimbriae, at a mucosal surface or on a catheter or tube. It then resists host defenses: the capsule blocks phagocytosis and complement, and siderophores let it get the iron it needs to multiply. As it grows and dies, LPS drives inflammation, which produces the local damage (for example, in the lung) and, if the organism reaches the blood, the systemic picture of sepsis. In hypervirulent strains, the heavy capsule and extra siderophores allow spread to distant organs, giving the liver abscess and metastatic infections that make these strains distinctive.

This is also why resistance matters so much here. A Klebsiella strain that combines these virulence factors with ESBL or carbapenemase production is both hard to clear and hard to treat, which is the combination that makes the organism a global priority pathogen.

Identifying Klebsiella pneumoniae in the laboratory

Specimen. Sputum, mid-stream urine, or blood, depending on the suspected infection. Classic currant-jelly sputum may be seen in Klebsiella pneumonia, though it is not common.

On the plate.

• Blood agar: mucoid, non-hemolytic colonies.
• MacConkey agar: mucoid, pink (lactose-fermenting) colonies.

The mucoid pink colony is the first-look signature. The tests below confirm it.

The identification panel

No single test identifies Klebsiella. The pattern does. Each test below states the Klebsiella result, what it means, and what a different result would tell you. For the full method and reading of any one test, follow its link.

How to read the IMViC block quickly. Klebsiella pneumoniae is IMViC − − + + (indole−, MR−, VP+, citrate+). The organism it is most often contrasted with, E. coli, is the mirror image, + + − −. If you remember one contrast for this family, remember that one.

For the full step-by-step method, reagents, and reading of each test, use the linked test pages above. This page gives the Klebsiella result and what it means; the test pages give the procedure.

Confirming and separating from look-alikes

Many laboratories do not run every tube. A common shortcut is TSI, an indole/motility medium (SIM or similar), and citrate, which together sort most Enterobacteriaceae. When results are unclear, a full commercial panel such as API 20E or Enterotube is used.

The two separations students most need:

• From E. coli: Klebsiella is non-motile, mucoid, VP-positive, indole-negative. E. coli is motile, dry, VP-negative, indole-positive. They sit on opposite ends of IMViC.
• From Klebsiella oxytoca: the two are very similar, but K. oxytoca is indole-positive and K. pneumoniae is indole-negative. Indole is the deciding test.

Resistance: why Klebsiella pneumoniae is a priority pathogen

Klebsiella pneumoniae is one of the most important carriers of resistance in Gram-negative bacteria. Two mechanisms matter most. It is a frequent producer of extended-spectrum beta-lactamases (ESBLs), which destroy most cephalosporins. And it is the source of the most common carbapenemase, Klebsiella pneumoniae carbapenemase (KPC), which is why carbapenem-resistant Enterobacteriaceae are such a concern. Detection and mechanism are covered in the beta-lactamase and carbapenem-resistance articles.

How to remember

The mucoid string. Picture touching a Klebsiella colony with a loop and pulling up a long, sticky string of capsule. That one image carries three facts at once: the thick capsule, the mucoid wet colonies, and the reason the organism resists your immune cells. Ask yourself: why does this colony look so wet and glistening? Capsule. Same answer explains its virulence.

Non-motile is the odd one out. Most of this family swims; Klebsiella sits still. Tie it together: no movement, no flagella, no H antigen. Three "no"s that travel as a set. If a question tells you an Enterobacteriaceae isolate is non-motile and mucoid, Klebsiella should be your first thought.

IMViC mirror. Klebsiella is − − + +, E. coli is + + − −. They are opposites. Remember one and you have the other by flipping every sign. Self-check: is Klebsiella indole-positive? No, that is E. coli (and K. oxytoca).

Key exam facts in one table

Where students get confused

Oxidase result read the wrong way. Klebsiella is oxidase-negative. A common error is running a full enteric panel on an oxidase-positive rod. If oxidase is positive, stop: you are likely looking at Pseudomonas, not Klebsiella.

Assuming lactose fermentation is unique. Pink on MacConkey narrows the field but does not confirm Klebsiella. E. coli, Enterobacter, and Citrobacter are also pink. The mucoid texture plus non-motility plus IMViC is what confirms it, not the color alone.

Klebsiella pneumoniae vs Klebsiella oxytoca. These get mixed up constantly because they are nearly identical. The single deciding test is indole: K. oxytoca positive, K. pneumoniae negative.

TSI misread as H₂S-positive. Klebsiella produces strong gas (the butt cracks and lifts), which students sometimes confuse with the black color of H₂S. Klebsiella does not make H₂S. Gas lifts the medium; H₂S turns it black. Different readings.

Urease overcalled. Klebsiella is urease-positive, but weakly and slowly. Do not read it as a strong rapid positive and jump to Proteus, which splits urea fast and hard.

Currant-jelly sputum treated as always present. It is the classic teaching image but is uncommon in practice. Its absence does not rule Klebsiella out.

Capsule confused with the string test. All Klebsiella have a capsule and look mucoid. The string test is a further step: it flags the hypervirulent strains that make an unusually heavy capsule. A mucoid colony is normal for the species; a long string (>5 mm) points to a hypervirulent strain.

References

• Tille, P. M. (2022). Bailey & Scott's Diagnostic Microbiology (15th ed.). Elsevier.
• Ashurst, J. V., & Dawson, A. (2023). Klebsiella pneumonia. In StatPearls. StatPearls Publishing. https://www.ncbi.nlm.nih.gov/books/NBK519004/
• Qu, T. T., Zhou, J. C., Jiang, Y., Shi, K. R., Li, B., Shen, P., Wei, Z. Q., & Yu, Y. S. (2015). Clinical and microbiological characteristics of Klebsiella pneumoniae liver abscess in East China. BMC Infectious Diseases, 15, 161. https://doi.org/10.1186/s12879-015-0899-7
• Madigan, M. T., Bender, K. S., Buckley, D. H., Sattley, W. M., & Stahl, D. A. (2021). Brock Biology of Microorganisms (16th ed.). Pearson.