Escherichia coli: Identification, Biochemical Tests, and Disease

A woman comes to the clinic with a burning feeling when she urinates and a frequent urge to go. The clinic sends a urine sample to the laboratory. By the next morning, one organism has grown, and it is the most likely cause: Escherichia coli. It is behind more than three out of four urinary tract infections.

E. coli is strange in this way. It lives quietly in everyone's gut and usually causes no harm. Yet it is also the most common cause of urinary tract infection in the world, and some strains cause severe diarrhea. This page is about telling E. coli apart in the laboratory, and understanding why the same organism can be both harmless and dangerous.

Overview

Escherichia coli (written E. coli) is a Gram-negative rod of the family Enterobacteriaceae. It lives as normal flora in the gut of humans and warm-blooded animals. Most strains are harmless, and some even help the host by making vitamin K. A minority of strains have picked up virulence factors and can cause disease, either in the gut or outside it.

This is the key idea to hold onto: E. coli is not one thing. Whether a strain is harmless or harmful depends on which virulence factors it carries. The strains that cause urinary infection are different from the ones that cause diarrhea, and both are different from the gut commensal.

Diseases caused by E. coli

Figure: Different strains of Escherichia coli and diseases caused by them

• Urinary tract infection (UTI). This is the headline. E. coli is the most common cause of both community-acquired and hospital-acquired UTI, causing more than 75% of cases. The strains responsible are called uropathogenic E. coli (UPEC), covered in detail below.
• Bloodstream infection (sepsis). E. coli is one of the leading causes of Gram-negative sepsis, often starting from a urinary or abdominal source.
• Neonatal meningitis. A newborn can be exposed to E. coli in the birth canal during delivery. Certain strains (especially those with the K1 capsule) are a leading cause of meningitis in the newborn.
• Diarrhea. Several distinct strains cause intestinal disease, from watery traveler's diarrhea to bloody diarrhea with a risk of kidney failure. Because these strains work by different mechanisms, they are covered in a separate article: Diarrheagenic Escherichia coli.

What E. coli looks like, and what that tells you

Escherichia coli is a Gram-negative short rod and a facultative anaerobe. It is the most abundant facultative anaerobe in the colon. A few features do the identification work.

Figure: LF and NLF colonies in MacConkey Agar

It ferments lactose, so it is pink on MacConkey agar. This is the single most useful early clue and the most searched fact about this organism. On MacConkey agar, E. coli forms pink, shiny colonies about 0.5 to 1 mm across after overnight growth. This pink color separates it from Salmonella and Shigella, the two intestinal pathogens you most want to catch, which stay pale because they do not ferment lactose. So a pink colony in a stool culture is usually not the pathogen; a pale one is the suspect.

On EMB agar, it gives a green metallic sheen. This is the E. coli signature. The strong acid it produces from fermenting lactose makes the dye in the medium precipitate on the colony surface, which reflects light as a green metallic shine. When you see that green sheen, think E. coli first. Other lactose fermenters such as Enterobacter grow pink on EMB but usually lack the sheen.

It is motile. Unlike Klebsiella, which sits still, E. coli swims. This is one quick way to separate the two pink, lactose-fermenting look-alikes.

One trap: not every E. coli is a lactose fermenter, and one dangerous strain has a special twist. A few E. coli (the Alkalescens-Dispar group) do not ferment lactose and look pale. More importantly, E. coli O157:H7, the strain that causes bloody diarrhea, does not ferment sorbitol. Because ordinary E. coli does ferment sorbitol, sorbitol-MacConkey agar makes O157:H7 stand out as a pale colony among pink ones. This is the basis of screening for it. The full O157 story is in the diarrheagenic E. coli article.

Surface antigens

E. coli has over 1,000 antigenic types, which is why strains are named by their antigen formula (for example, O157:H7). Three antigens are used:

• O antigen (part of the LPS in the cell wall): over 150 types.
• H antigen (the flagellar protein): over 50 types.
• K antigen (the capsule): over 90 types. The K1 capsule type is the one linked to neonatal meningitis.

Virulence factors and how E. coli causes disease

A commensal E. coli and a uropathogenic one look almost identical on a plate. What separates them is a set of virulence factors that let the harmful strain stick, survive, and damage tissue where it does not belong. Each factor below is tied to what it does in the body.

Adhesins (fimbriae/pili). Before E. coli can cause infection, it has to hold on. In the urinary tract, urine flow constantly flushes organisms out. P fimbriae and type 1 fimbriae let uropathogenic E. coli grip the lining of the urethra and bladder so it is not washed away. Attachment is the necessary first step; without it, there is no infection. In gut strains, different fimbriae grip the intestinal lining instead.

Capsule (K antigen). The polysaccharide capsule coats the organism and blocks phagocytosis and complement, so white blood cells and serum cannot kill it easily. This is what lets E. coli survive in the blood and cause sepsis. The K1 capsule type is especially important: it helps the organism resist host defenses and is the type most linked to neonatal meningitis.

Endotoxin (lipopolysaccharide, LPS). LPS is the classic Gram-negative endotoxin, part of the outer membrane. When E. coli reaches the blood, LPS triggers a strong inflammatory response. In severe infection this produces the fever, low blood pressure, and, in the worst cases, disseminated intravascular coagulation (DIC) of Gram-negative sepsis.

Hemolysin. This toxin punches holes in host cell membranes, including red blood cells. It damages tissue in the urinary tract and helps the organism free up iron and nutrients from the cells it destroys.

Aerobactin (siderophore). Iron is scarce inside the body because the host locks it away. Aerobactin is a molecule E. coli releases to scavenge iron from host proteins, so it can grow where iron is limited. Uropathogenic strains that make aerobactin have a growth advantage in the urinary tract.

How uropathogenic E. coli causes a UTI

The sequence explains why UPEC is so good at causing urinary infection. The organism first reaches the urethra, usually from the gut, since the two openings are close. It then attaches using P and type 1 fimbriae, so urine flow cannot flush it out. It climbs toward the bladder and sometimes the kidney. Along the way the capsule protects it from immune attack, hemolysin damages the tissue lining, and aerobactin scavenges iron so it can keep multiplying. If the organism then reaches the blood, LPS drives the systemic inflammation of sepsis. The more of these factors a strain carries, the more severe the infection it can cause.

This is also why the same species can be harmless in the gut and dangerous in the bladder: the commensal strain simply lacks most of these tools.

Identifying E. coli in the laboratory

Specimen. Urine (for UTI, the most common request), blood, or stool, depending on the suspected infection.

Figure: Scheme for Rapid Identification of E. coli

On the plate.

• MacConkey agar: pink, shiny colonies, 0.5 to 1 mm.
• EMB agar: colonies with a green metallic sheen (the E. coli signature).
• Blood agar: grey to white colonies, variable; some strains are hemolytic.

The identification panel

No single test identifies E. coli. The pattern does. Each test below gives the result and what it means. For the full method of any one test, follow its link.

Read the IMViC block as one pattern. E. coli is IMViC + + − − (indole+, MR+, VP−, citrate−). This is the classic teaching contrast with Klebsiella/Enterobacter, which are the mirror image, − − + +. If you remember one contrast for this whole family, remember that one: the gut commensal E. coli splits tryptophan and pours out acid; the environmental organisms use citrate instead.

For the full step-by-step method of each test, use the linked pages above. This page gives the E. coli result and what it means.

Separating E. coli from its look-alikes

• From Klebsiella: E. coli is motile, indole-positive, dry colonies, IMViC + + − −. Klebsiella is non-motile, indole-negative, mucoid colonies, IMViC − − + +. They are opposites on almost every test.
• From Salmonella and Shigella: E. coli ferments lactose (pink on MacConkey); these two do not (pale). This is the first sort in a stool culture.

Antimicrobial resistance in E. coli

E. coli is one of the most important carriers of antibiotic resistance worldwide. The main concern is the extended-spectrum beta-lactamase (ESBL), a plasmid-carried enzyme that destroys most cephalosporins and aztreonam but is blocked by clavulanic acid. Rising cephalosporin resistance in E. coli is mostly due to ESBLs, which usually evolved by point mutations of the older TEM-1 and SHV-1 enzymes. Some strains go further and become carbapenem-resistant, which leaves very few treatment options.

In the laboratory, ESBL production is screened by reduced susceptibility to third-generation cephalosporins and confirmed by showing that clavulanic acid restores the zone of inhibition. The full detection methods (combination disc and double disc synergy) and the CLSI interpretive cutoffs are covered in the beta-lactamase and ESBL article.

How to remember

The green sheen means E. coli. Picture a colony on EMB agar shining like a beetle's back, a green metallic shine. That shine is strong acid from lactose fermentation. When you see it, think E. coli. Ask yourself: what makes the sheen? Acid from lactose. Same fact, one image.

IMViC mirror. E. coli is + + − −, Klebsiella is − − + +. They are exact opposites. Learn one and flip every sign to get the other. Self-check: is E. coli citrate-positive? No. That is the Klebsiella side. E. coli is the acid-maker, not the citrate-user.

Pink but motile. Both E. coli and Klebsiella are pink on MacConkey. The quick separators: E. coli moves and is indole-positive with dry colonies; Klebsiella sits still and is indole-negative with mucoid colonies. Motility plus indole tells the two apart.

Fimbriae first. For UTI, the whole story starts with grip. Picture urine trying to flush the organism out and the fimbriae holding on. If it cannot attach, it cannot infect. That one image anchors why adhesins are the first virulence factor that matters.

Key exam facts

Where students get confused

Treating all E. coli as lactose fermenters. Most are, and are pink on MacConkey, but the Alkalescens-Dispar group is not, and O157:H7 is a sorbitol non-fermenter. "Lactose fermenter" is a strong clue, not a guarantee.

E. coli vs Klebsiella on a pink plate. Both are pink lactose fermenters, so color alone will not separate them. The fast separators are motility (E. coli moves, Klebsiella does not), indole (E. coli positive, Klebsiella negative), and colony texture (E. coli dry, Klebsiella mucoid).

Confusing the two kinds of E. coli disease. The strains that cause UTI (uropathogenic E. coli) are not the same as the strains that cause diarrhea. They carry different virulence factors and are studied separately. Do not mix UPEC virulence factors with the intestinal toxin mechanisms.

MUG and O157. MUG is a useful rapid E. coli test, but the one strain you most need to catch, O157:H7, is MUG-negative and sorbitol-negative. The rapid tests that work for ordinary E. coli are exactly the ones that miss O157. That reversal is a common exam trap.

Green sheen is not universal. The EMB green sheen strongly suggests E. coli, but not every lactose fermenter shows it, and occasional E. coli strains show it weakly. Use it as a strong pointer, then confirm with biochemistry.

References

1. Tille, P. M. (2022). Bailey & Scott's Diagnostic Microbiology (15th ed.). Elsevier.
2. Procop, G. W., et al. (2017). Koneman's Color Atlas and Textbook of Diagnostic Microbiology (7th ed.). Wolters Kluwer.
3. Levinson, W., Chin-Hong, P., Joyce, E. A., Nussbaum, J., & Schwartz, B. (2020). Review of Medical Microbiology and Immunology: A Guide to Clinical Infectious Diseases (16th ed.). McGraw Hill.
4. Madigan, M. T., Bender, K. S., Buckley, D. H., Sattley, W. M., & Stahl, D. A. (2021). Brock Biology of Microorganisms (16th ed.). Pearson.