Streptococci - Overview of Detection, Identification, Differentiation and Cultivation Techniques

Jvo Siegrist

AnalytiX Volume 7 Article 3

ivo.siegrist@sial.com

Streptococci are non-motile, microaerophilic, Grampositive spherical bacteria (cocci). They often occur as chains or pairs and are facultative or strict anaerobes. Streptococci give a negative catalase test, while staphylococci are catalase-positive. The cell division of Streptococci species involves two separate biosynthetic events: peripheral cell-wall elongation and septal-wall synthesis.¹ Because they are unable to synthesize cytochromes, Streptococci cannot carry out oxidative phosphorylation. They are able to ferment sugars, but the end product is always lactic acid. Therefore, Streptococci are very acid tolerant and count among the lactic acid bacteria order.

There are many natural sources of Streptococci, including humans and diverse animals where they often colonize the mucosal surfaces of mouth, intestinal tract, nasal passages and pharynx. The presence of Streptococci  in drinking water indicates fecal contamination. Food sources with high risk of Streptococcal contamination include milk and dairy products, eggs, steamed lobster, ground ham, potato salad, custard, rice pudding and shrimp salad. In most Streptococcal food poisoning cases, the food was allowed to stand at room temperature for several hours between preparation and consumption. The contamination of the food is most often the result of poor hygiene, handling of the food by infected people, or the use of raw (unpasteurized) milk. Although they can be potent pathogens, some Streptococci are commercially important for the production of cheese and yogurt. These include S. lactis, S. cremoris, S. diacelillactis and S. thermophilus, the latter being the most well-known.

For detection, identification, differentiation, enumeration and cultivation of Streptococci, we provide a broad range of specific agars and broths (Table 1), Streptococci Diagnostic Tests (Table 2) and a Gram staining kit and component solutions (Table 3).

Scientific classification of Streptococcus

Kingdom: Eubacteria
Phylum: Firmicutes
Class: Bacilli
Order: Lactobacillales
Family: Streptococcaceae
Genus: Streptococcus

Grouping of Streptococci based on hemolytic activity

Streptococci are divided into three groups based on their hemolytic (red blood cell lysing) activity. The hemolytic reaction can be visualized on blood agar plates, such as the non-selective Agars for Differentiation that are listed in Table 1.

Non-selective Enrichment Broths

Cat. No.	Brand	Description
53286	Sigma-Aldrich	Brain Heart Broth
B2551	Sigma-Aldrich	Brewer thioglycollate medium
22089	Sigma-Aldrich	Casein peptone Lecithin Polysorbate Broth
22098	Sigma-Aldrich	CASO Broth
60865	Sigma-Aldrich	Cooked Meat Broth
17123	Sigma-Aldrich	Elliker Broth
5121	Sigma-Aldrich	Heart Infusion Broth
70122	Sigma-Aldrich	Nutrient Broth No. 1
70149	Sigma-Aldrich	Nutrient Broth No. 3
40893	Sigma-Aldrich	Peptone Water, phosphate-buffered, Vegitone
70179	Sigma-Aldrich	Peptone Water
77187	Sigma-Aldrich	Peptone Water, phosphate-buffered
S4681	Sigma-Aldrich	Standard Nutrient Broth No. 1
85905	Sigma-Aldrich	Stuart Ringertz Medium
90404	Sigma-Aldrich	Thioglycolate Broth with Resazurine
T1438	Sigma-Aldrich	Todd Hewitt Broth
22092	Sigma-Aldrich	Tryptic Soy Broth
51228	Sigma-Aldrich	Tryptic Soy Broth No. 2
41298	Sigma-Aldrich	Tryptic Soy Broth, Vegitone
T3938	Sigma-Aldrich	Tryptone Soya Broth without Dextrose
T4407	Sigma-Aldrich	Tryptose Broth
V5262	Sigma-Aldrich	Veal Infusion Broth

Selective Enrichment Broths

Cat. No.	Brand	Description
17157	Sigma-Aldrich	Glucose Azide Broth

Non-selective Agars for Cultivation, Enumeration and Isolation

Cat. No.	Brand	Description
A3340	Sigma-Aldrich	AC Agar
70138	Sigma-Aldrich	Brain Heart Infusion Agar
22095	Sigma-Aldrich	CASO Agar
27688	Sigma-Aldrich	Columbia Agar
D3060	Sigma-Aldrich	Dextrose starch agar
D8184	Sigma-Aldrich	Diagnostic sensitivity test agar
70147	Sigma-Aldrich	Milk Agar
70148	Sigma-Aldrich	Nutrient Agar
17197	Sigma-Aldrich	Peptonized Milk Agar
70152	Sigma-Aldrich	Plate Count Agar
88588	Sigma-Aldrich	Plate Count Agar according to Buchbinder et al.
19718	Sigma-Aldrich	Plate Count Agar, Vegitone
17257	Sigma-Aldrich	Streptococcus thermophilus Isolation Agar
22091	Sigma-Aldrich	Tryptic Soy Agar
14432	Sigma-Aldrich	Tryptic Soy Agar, Vegitone
51414	Sigma-Aldrich	Tryptic Soya Agar with Polysorbate 80 and Lecithin
70159	Sigma-Aldrich	Tryptone Glucose Extract Agar
T2313	Sigma-Aldrich	Tryptose Agar

Non-selective Agars for Differentiation

Cat. No.	Brand	Description
70133	Sigma-Aldrich	Blood Agar (Base)
B1676	Sigma-Aldrich	Blood Agar Base No. 2
55420	Sigma-Aldrich	CLED Agar
70136	Sigma-Aldrich	Deoxyribonuclease Test Agar
D2560	Sigma-Aldrich	DNase Test Agar with Toluidine Blue
17153	Sigma-Aldrich	LS Differential Agar

Selective Agars with Differential System for Differentiation, Detection and Isolation

Cat. No.	Brand	Description
06105	Sigma-Aldrich	Bile Esculin Azide Agar
17151	Sigma-Aldrich	Kanamycin Esculin Azide Agar
01337	Sigma-Aldrich	Mitis Salivarius Agar

Media for Sensitivity Testing

Table 1 Media for Streptococci

Cat. No.	Brand	Description
97580	Sigma-Aldrich	Mueller-Hinton Agar 2

Alpha-Hemolytic Streptococci

In alpha hemolysis, the red blood cells remain intact, but the hemoglobin is converted to biliverdin. This causes dark greening of the blood agar plate around the colonies.

• Pneumococci
  S. pneumoniae: Causes bacterial pneumonia, otitis media and meningitis. S. pneumoniae sensitivity to optochin (ethyl hydrocupreine hydrochloride) is the basis of a diagnostic test (Figure 1, Table 2).

Figure 1. Differentiation and identification flow chart of Gram-positive cocci

Table 2 Diagnostic tests for identification and differentiation of streptococci

Cat. No.	Brand	Description
88597	Sigma-Aldrich	Catalase Test
08382	Sigma-Aldrich	Bacitracin Disks
74042	Sigma-Aldrich	Optochin Disks
67886	Sigma-Aldrich	PYRase Strips

• Viridans and Others
  S. mutans: Associated with dental caries.
  S. viridans: Causative agent for endocarditis and dental abscesses.
  S. salivarius: Considered to be an opportunistic pathogen.
  S. salivarius subsp. thermophilus: Used in the production of cheese and yogurt.

Beta-Hemolytic Streptococci

Beta hemolysis is a complete hemolysis of erythrocytes by the enzyme hemolysin. Clear zones will appear around the colonies on the blood agar plate. Beta-hemolytic Streptococci are further divided into serological groups using specific antibodies that recognize surface carbohydrate antigens (e.g. A, B, etc.).

• Group A
  S. pyogenes: Causes infections like strep throat, acute rheumatic fever, scarlet fever, acute glomerulonephritis and necrotizing fasciitis. It is sensitive to bacitracin (Fig. 1), a fact that is employed in a Streptococcus diagnostic test (Table 2). Other Streptococci species may also possess the Group A antigen but are not human pathogens. These include S. dysgalactiae subsp. equisimilis and S. anginosus strains.
• Group B
  S. agalactiae: Causes meningitis in neonates and the elderly. Occasionally colonizing the female reproductive tract, they increase the risk for premature rupture of membranes and transmission of the infection to the child.
• Group C
  S. equi: Causes strangles infection in horses.
  S. zooepidemicus: Causes infections in cattle, horses and other mammals.
• Group D (Enterococci)
  Many former Group D Streptococci have been reclassified to the genus Enterococcus (e.g. S. faecalis, S. faciem, S. durans, S avium).
  S. bovis and S. suis: Still in the Group D streptococci.

Non-Hemolytic or Gamma-Hemolytic Streptococci

Gamma hemolysis is a misnomer as there is actually no hemolysis. Non-hemolytic or gamma-hemolytic Streptococci rarely cause disease.

Table 3 Gram staining kit and component solutions

Cat. No.	Brand	Description
77730	Sigma-Aldrich	Gram Staining Kit
94448	Sigma-Aldrich	Gram‘s Crystal violet Solution
75482	Sigma-Aldrich	Gram‘s Decolorizer Solution
87794	Sigma-Aldrich	Gram’s Fuchsin Solution
90107	Sigma-Aldrich	Gram‘s Iodine Solution
94635	Sigma-Aldrich	Gram‘s Safranin Solution

References

1. Beilharz K, Novakova L, Fadda D, Branny P, Massidda O, Veening J. 2012. Control of cell division in Streptococcus pneumoniae by the conserved Ser/Thr protein kinase StkP. Proceedings of the National Academy of Sciences. 109(15):E905-E913. https://doi.org/10.1073/pnas.1119172109