Lancefield Classification of Streptococci

When a laboratory grows a beta-hemolytic streptococcus, the first question is which group it belongs to, because the group often predicts the disease and guides treatment. This page explains how streptococci are grouped and introduces the quick bench tests to identify each group.

Streptococci are Gram-positive cocci either diplococci (lanceolate shaped; S. pneumoniae) or in chains. They are catalase-negative (an important test to differentiate streptococci from Staphylococci). These fastidious gram-positive cocci may give either alpha or beta or gamma hemolysis when cultured on blood agar.

β-hemolytic streptococci produce hemolysins that lyse the sheep RBCs (poorly lyse human RBCs too), resulting in a clearing of the Blood Agar plate surrounding the colonies. More intense β-hemolysis is noted in areas where the medium has been “stabbed,” pushing some of the bacteria under the surface of the medium. The β-hemolysis in these areas is due to the combined action of both hemolysins of Streptococci (Streptolysin O and Streptolysin S). The surface β-hemolysis is largely due to streptolysin S (oxygen stable hemolysin), as Streptolysin O which is oxygen-labile does not show maximal activity on the surface of the agar.

Figure: Beta Hemolysis in Sheep Blood Agar.

Serologic grouping of cell wall carbohydrates of Streptococci was classically been used to identify species of beta-hemolytic Streptococci, this grouping is known as  Lancefield grouping. Beta-hemolytic streptococci are arranged into twenty groups, A to V excluding I and J (that is, A through H and K through V). Rebecca Lancefield, a prominent American microbiologist, introduced the system. Of these twenty, only a few commonly infect humans, mainly groups A, B, C, D, F, and G, and this page focuses on those.

Figure: Streptococcus as seen in scanning electron microscope

The basis of Lancefield grouping is the antigenic differences in C carbohydrates, a group-specific antigen.  C carbohydrate is located in the cell wall of streptococci

(Note: viridans streptococci and Streptococcus pneumoniae have no group-specific antigen).

The original Lancefield precipitin test is now rarely performed in clinical laboratories. Now, it has been replaced by either latex agglutination or coagglutination.

Group A Streptococci (GAS)

Streptococcus pyogenes, is one of the most important human pathogens which causes Streptococcal sore throat, skin infections, and post-streptococcal sequelae such as rheumatic fever and post-streptococcal glomerulonephritis (PSGN). S. pyogenes is inhibited by the antibiotic bacitracin, an important diagnostic criterion.

Group B Streptococci

Streptococcus agalactiae colonizes the genital tract of some women and can cause neonatal meningitis and sepsis. It can cause septic abortion and puerperal or gynecological sepsis and occasionally urinary tract infections. S. agalactiae gives CAMP test positive and can hydrolyze the hippurate.

Group C Streptococci

Group C beta-hemolytic streptococci currently include S. dysgalactiae subsp. equisimilis and other related strains under the same species or S. equi subspecies. S. dysgalactiae subsp. equisimilis has been recovered from pharyngitis and tonsillitis from patients with underlying diseases such as cardiopulmonary disease, diabetes, immunosuppression, HIV, etc.

Group D Streptococci

It includes enterococci (e.g., Enterococcus faecalis and Enterococcus faecium) and nonenterococci (S. bovis). Enterococci are members of the normal flora of the colon. They can cause urinary, biliary, and cardiovascular infections.

Note: By the mid-1980s, the enterococcal streptococci (Lancefield group D) had taken up residence in their own newly created Enterococcus genus and the “dairy” or “lactic” streptococci (Lancefield group N) were moved to new Lactococcus genus.

Group F Beta Hemolytic streptococci

Organisms of this group have been called S. milleri in the British taxonomic scheme and anginosus (S. anginosus) group in the American taxonomic group.  Group F Beta-hemolytic streptococci are recognized as a cause of severe suppurative infections including cellulitis, deep-tissue abscesses, bacteremia, osteomyelitis, and endocarditis.

Other groups (K to U): These contain streptococcal species of limited virulence that can cause infections in immunocompromised individuals.

Summary of Medically Important Streptococci

Where students actually get confused

• S. pneumoniae and viridans streptococci don't get a Lancefield letter, full stop. They lack the C-carbohydrate antigen entirely. Don't try to assign them a group, the correct answer is "not groupable," and that's exactly why optochin and bile solubility exist as a separate identification path.
• "Group D" is a historical convenience, not current taxonomy. Enterococcus was reclassified out of genus Streptococcus into its own genus in the mid-1980s. Exam questions and older texts still group Enterococcus and S. bovis together as "Group D" for teaching purposes, but they're genetically distinct genera now, separated at the bench by salt tolerance.
• Group C and G aren't always bacitracin-resistant. As covered in the Bacitracin Test article, occasional Group C and G isolates show susceptibility, which is exactly why PYR exists as the tie-breaker for confirming true GAS.

How to Remember

This page ties together the individual streptococcal identification tests. Each of those tests, bacitracin, CAMP, PYR, salt tolerance, bile solubility, and optochin, exists because serological grouping is not practical for a routine laboratory.

The summary table above pulls all of these tests together: GAS is bacitracin-sensitive and PYR-positive; GBS reverses both and adds CAMP-positive and hippurate-positive; Group D splits on salt tolerance alone; and S. pneumoniae and the viridans group are not groupable at all. Those two are told apart by bile solubility and optochin instead of serology, because they do not carry the C-carbohydrate antigen this whole system depends on.

References

1. Procop, G. W., et al. (2017). Koneman's Color Atlas and Textbook of Diagnostic Microbiology (7th ed.). Wolters Kluwer.
2. Tille, P. M. (2022). Bailey & Scott's Diagnostic Microbiology (15th ed.). Elsevier.
3. Şahan Yapicier, Ö., Sababoglu, E., Ozturk, D., Turutoglu, H., Pehlivanoglu, F., & Kaya, M. (2021). Lancefield classification and antimicrobial resistance of hemolytic streptococci isolated from bovine mastitis. Veterinaria italiana, 57(1), 41–47. https://doi.org/10.12834/VetIt.1855.9879.3 
4. Facklam, R. F., Martin, D. R., Lovgren, M., Johnson, D. R., Efstratiou, A., Thompson, T. A., Gowan, S., Kriz, P., Tyrrell, G. J., Kaplan, E., & Beall, B. (2002). Extension of the Lancefield classification for group A streptococci by addition of 22 new M protein gene sequence types from clinical isolates: emm103 to emm124. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America, 34(1), 28–38. https://doi.org/10.1086/324621