Gram Positive Bacteria
MICROBIOLOGY
Dr. Pramila Singh
2/22/20248 min read
Gram Positive Bacteria
Staphylococci
Streptococci and Pneumococci
Enterobacteriacae - (Ecoli, Salmonella, Shigella).
STAPHYLOCOCCI
1. General characteristics of Staphylococci:
i. Gram-positive cocci
ii. They are found in regular or irregular cluster
iii. White, grey, or golden yellow colonies
iv. They are aerobic bacteria,
v. Nonmotile, non-spore-forming, and non-capsulated bacteria
vi. Parasite as a part of bacterial flora of human skin, upper respiratory tract, intestinal tract
vii. Grow in carbohydrate-rich culture media
viii. Ferment carbohydrates without release of gas,
2. Morphology:
i. Shape: Round (Cocci)
ii. Size: 0.5 to 1 µm in diameter
iii. Cells arrangement: Grapes like cluster. This characteristic distinguishes Streptococci from staphylococci. Streptococci form a chain-like structure, not a grape-like structure.
iv. Non-motile, non-spore forming, and non-capsulated, Some may release a slight amount of capsulate materials.
1. Staining: Same as discussed under gram-positive bacteria staining.
2. Culture: They can grow in all ordinary culture media such as Nutrient agar media, blood agar media, Milk agar media, Mannitol salt agar media, and Liquid culture medium. The optimum pH for growth is 7.4 to 7.6. The optimum temperature for growth is 10 to 42 degrees C. They can tolerate 8 to 10 percent salt concentration. They are aerobic and facultative anaerobic bacteria.
Cultural characteristics: S. aureus: As per the above table.
3. Biochemical Characteristics: They release several biochemical such as
i. Endotoxin: It causes RBC hemolysis
ii. Leucocidin: It kills WBCs.
iii. Enterotoxin: It causes vomiting and diarrhea
iv. Coagulase: It causes blood clotting by converting fibrinogen to fibrin.
v. Staphylokinase: It causes fibrinolysis
vi. Penicillinase: It inactivates antibiotic penicillin.
vii. Biochemical reactions
6. Antibiotics related to Staphylococci: Originally, they were sensitive to the antibiotic penicillin. Now they have developed resistance to penicillin. They produce the enzyme penicillinase that inactivates the antibiotic penicillin. They are sensitive to other antibiotics such as erythromycin, cloxacillin, rifampicin, cephalosporin, etc.
7. Distribution: Parasite is a part of the bacterial flora of human skin, upper respiratory tract, and intestinal tract in healthy and infected people. Infected handkerchiefs, bed linen, blankets, infected domestic animals.
4 STREPTOCOCCI
1. General characteristics of Streptococci:
i. Gram-positive cocci. They may become gram-negative on aging and after death.
ii. Spherical in shape 0.5 to 1 µm diameter. They are arranged in pairs, short chain or long chain. Their chain length in liquid culture media is longer than in solid culture media.
iii. Heterogeneous group of bacteria. There are several types of streptococci depending upon their hemolysis ability:
a. α-hemolytic streptococci: Causes partial hemolysis in blood agar media. Ex.
b. β- hemolytic streptococci: Causes complete hemolysis in blood agar media. They are further divided into various groups:
· Group-A: S. pyogen
· Group-B: S. agalactiae
· Group C and G
· Group-D
· Group E, F, H, K, and U.
c. γ- hemolytic streptococci or non-hemolytic streptococci: No change in blood agar media.
iv. Some species of streptococci have capsules made of hyaluronic acid or polysaccharides.
v. White or grey colonies,
vi. They are facultative anaerobic bacteria,
vii. Non motile, non spore forming,
viii. They are non-halophilic but can tolerate up to 6.5% sodium chloride concentration.
ix. Catalase test negative. This test differentiates Streptococci from staphylococci. Staphylococci show catalase test positive.
x. They ferment most of the sugar to produce acid without the release of gas.
2. Morphology of Streptococci: Spherical in shape 0.5 to 1 µm diameter. They are arranged in pairs, short chain or long chain. Their chain length in liquid culture media is longer than in solid culture media. They develop white or grey colonies with irregular outlines. Normally diameter of the colony remains less than 1mm. A colony of virulent streptococci has a matt appearance. A colony of nonvirulent streptococci has a glossy appearance. Some species of streptococci have capsules made of hyaluronic acid or polysaccharides. They are nonmotile, non-spore-forming bacteria.
3. Staining of Streptococci: Same as discussed in gram-positive bacteria staining.
4. Culture of Streptococci: They are facultative anaerobe or aerobic bacteria. The optimum temperature for growth is 370 C. Blood agar media is the most suitable culture media for its growth. 10% carbon dioxide in culture media promotes the growth of streptococci and hemolysis. They form small circular, disc-shaped, pinpoint, colorless glossy, opaque, convex colonies with clear hemolytic zones around colonies. Colonies develop within 24 hours at 370 C.
5. Biochemical characteristics of Streptococci: S. pyogenes several enzymes and exotoxins. Such as
i. Haemolysins: Haemolysis of RBC
ii. Streptokinase: Breakdown of fibrin around the wound.
iii. Hyaluronidase: Break hyaluronic acid in cells. Spread infection into intracellular space.
iv. Deoxyribonuclease: Liquify thick pus.
Catalase test: Negative
Bacitracin: Positive
Optochin: Negative
Bile solubility: Negative
5. Antibiotics related to Streptococci: Very sensitive to erythromycin, cotrimoxazole, and penicillin.
Distribution of Streptococci: The normal habitat of streptococci is milk, milk products, dust, vegetables, and water. Group A, C, and G streptococci are present in the human upper respiratory tract. Group B streptococci are present in human female genital organs. Group D Streptococci are present in the human small intestine.
PNEUMOCOCCI
(Pneumococci pneumoniae)
1. General characteristics of Pneumococci:
i. Lancet-shaped Gram-positive cocci
ii. Capsulated cocci
iii. Present in pair (diplococcus) or in short chain
iv. Aerobic or facultative anaerobic cocci
v. Optimum temperature of growth 37 degrees C.
vi. Optimum pH for growth is 7.8
vii. Catalase negative and oxidase negative cocci.
2. Morphology of Pneumococci: Natural occurring Pneumococci are flame-shaped or lanceolate-shaped cocci. They occur in pairs (diplococci) or short chains. Diplococci or short-chain cocci are covered in a capsule. The capsule is broader at one end and narrow at another end.
Pneumococci in artificial culture media do not have capsules.
3. Staining of Escherichia coli:: Same as discussed under Gram-positive bacteria staining.
4. Culture of Pneumococci: They require enriched culture media for their optimum growth. Chocolate agar media and blood agar media are suitable culture media. But chocolate agar media with 5 to 10% carbon dioxide is more suitable for Pneumococci growth.
Pneumococci form dome-shaped colonies in blood agar media after 18 hours of incubation. Colonies have around 0.5 to 1 mm diameter. The colony has greenish decolorisation around the colony. The colony becomes flat after 48 hours with a raised edge. It is due to the autolysis of Pneumococci cells in the culture media. Some develop large mucoid colonies under anaerobic incubation.
Pneumococci develop turbidity in liquid culture media enriched in glucose. But Pneumococci undergo autolysis.
1. Biochemical characteristics of Pneumococci:
Quelling test: Positive
Bile solubility: Positive
Inulin fermentation: Positive
Optochin sensitivity: Positive
Pneumococci ferment inulin to form acid. This test differentiates
2. Antibiotics related to Pneumococci: Sensitive to penicillin, erythromycin, and cotrimoxazole. Some are penicillin-resistant.
3. Distribution of Pneumococci: Human Upper respiratory tract.
Enterobacteriaceae - (Ecoli, Salmonella, Shigella)
Enterobacteria belong to Enterobacteriaceae. Enterobacteria are Gram-negative rods, Facultative anaerobic, Most of them are motile only a few are nonmotile, Non-sporing bacteria, Non acid-fast bacteria, and Grow in ordinary culture media over a wide range of temperatures. Ferment carbohydrate with the release of gas, Reduce nitrate to nitrite, Catalase test positive and Oxidase test negative. They are classified into three groups depending on their fermentation ability.
1. Lactose fermentation: Escherichia
2. Late lactose fermentation: Shigella sonnei
3. Non-Lactose fermentation: Salmonella, Shigella.
Escherichia coli (E. coli)
1. General characteristics of Enterobacteria:
i. Gram-negative straight rod arranged singly or in pairs.
ii. Peritrichous flagella make them motile, only a few are non-motile strains,
iii. Capsulated bacteria with fimbriae but no spore.
i. Ferment glucose, sucrose, maltose, mannitol, xylose, and lactose to form acid with the release of gas,
ii. Indole test positive, Methyl red test positive,
iii. Voges-Proskauer test negative, gelatine liquefication test negative, citrate test negative, urease test negative,
iv. Few strains are β hemolytic in blood agar culture media
v. Variation occurs in the colony from smooth to rough, especially on repeated culture.
vi. Pathogenicity: Diarrhoea, Urinary tract infection (UTI), Wound infection, Septicemia.
Morphology of Escherichia coli: Gram-negative straight rod-shaped bacteria arranged singly or in pairs. They have Peritrichous flagella that make them motile, only a few are non-motile strains. They are capsulated bacteria with fimbriae but they do not form spores. They develop pink-colored, circular-shaped, convex colony diameter 1 to 3 mm. Colony has an entire margin, opaque opacity with soft consistency.
Dr Pramila Singh
3. Staining of Escherichia coli: Same as discussed in gram-positive bacteria staining.
4. Culture of Escherichia coli: Nutrient agar, MacConkey’s agar, or Eosin-methylene blue agar are suitable culture media for the growth and isolation of E. coli.
Suitable biochemical media are glucose phosphate broth, motility agar, nutrient gelatine media, nitrate broth, sugars (glucose, sucrose, maltose, xylose, lactose, mannitol), etc.
The optimum incubation temperature is 370C, and the incubation period is 24 hours. E. coli develop smooth, soft, opaque, convex circular, 1 to 3 mm diameter colonies in culture media. Some strains are β haemolytic and some may develop mucoid colonies.
Biochemical characteristics of Escherichia coli: It ferments glucose, sucrose, maltose, mannitol, xylose, and lactose to form acid with the release of gas. It shows Indole test positive, Methyl red test positive, Voges-Proskauer test negative, gelatine liquefaction test negative, citrate test negative, and urease test negative.
Fermentation Reaction in Sugar
Glucose: Acid + Gas
Sucrose: Acid + Gas
Maltos: Acid + Gas
Mannitol: Acid + Gas
Xylos: Acid + Gas
Lactose: Acid + Gas
Biochemical reaction
Indole test positive: Positive,
Methyl red test: Positive
Nitrate test: Positive
Oxidase test: Negative
Urease test: Negative
Citrate test: Negative
Gelatine liquefaction test: Negative
Voges-Proskauer test: Negative
Antibiotics related to Escherichia coli: E. coli is sensitive to aminoglycosides antibiotics, cephalosporins, ampicillin, tetracyclines, nalidixic acid, nitrofurantoin, cotrimoxazole, etc
Distribution of Escherichia coli: Part of normal bacterial flora inside human intestine and animal intestine. They are also present in vegetables, water, and soil.
Salmonella
Salmonella is enterobacteria. Salmonella genus of bacteria includes several species. Among them three Salmonella species are most common parasite in the human body. These are Salmonella typhi, Salmonella suis, Salmonella enteritidis. Salmonella can also be divided into two groups depending upon their disease causing nature. These are Salmonella causing enteric fever and Salmonella causing food poisoning.
1. General characteristics of Salmonella:
i. Gram negative, Rod shaped motile bacilli
ii. Facultative anaerobic bacteria
iii. Ferment carbohydrate
iv. Oxidase test negative
v. H2S test Positive. KCN test positive, Citrate test positive
vi. Habitat: Human intestine, several vegetables
vii. Pathogenicity:
· S.typhi: Typhoid fever (Enteric fever), Nephrotyphoid, Osteomyelitis, spleen and liver abscess.
· S. paratyphi A and B: Paratyphoid fever, gastroenteritis.
· S. paratyphi C: Septicemia, Paratyphoid fever, arthritis, swelling in gall bladder.
Morphology of Salmonella: They are non capsulated rod shaped bacteria. They are arranged singly or in pairs. They have peritrichous flagella. They develop circular, soft, odourless colony, diameter 1 to 2 mm with irregular margin in culture media. Their opacity depends upon culture media that may be either translucent or opaque. Similarly colony elevation may be convex, raised andflat .
2. Culture of Salmonella: They readily grow in ordinary culture media such as nutrient agar media (NAM). Following combination culture media are preferred for Salmonella isolation: MacConkey agar media, Salanite F media, XLD (Xylose Lysine Deoxycholate) media, Deoxycholate citrate agar (DCA) media, Salmonella-Shigella agar media. The optimum temperature is 370 C and pH is 6.5 to 7.5 for optimum growth.
3. Biochemical characteristics of Salmonella: They ferment glucose, mannitol, and dulcite with the release of acid and gas. They do not ferment lactose sucrose, and salicin.
Antibiotics related to Salmonella: They are sensitive to Ampicillin, Cotrimoxazole, and chloramphenicol. However, most Salmonella species have developed resistance to chloramphenicol.
Distribution of Salmonella: They are parasites of the human body, animals, and birds. Birds such as hens, duck, etc, Animals such as cows, pigs goats, etc. S. typhi and S. paratyphi are parasites of humans only present in the intestine
Shigella
Four species of shigella cause bacillary dysentery in humans. These are S. dysenteriae, S. flexneri, S. boydii and S. sonnei.
1. General characteristics of Shigella:
i. Gram-negative, Rod-shaped, non-motile bacilli
ii. Aerobic and Facultative anaerobic bacteria
iii. Mostly ferment carbohydrates to release acid & no gas
iv. Mostly catalase negative, oxidase negative, citrate negative, and KCN negative.
v. Habitat: Human intestine
vi. Pathogenicity: Bacillary dysentery
2. Morphology of Shigella: They are rod-shaped bacteria that form colorless, transparent, odorless, soft, flat, circular colonies with an entire margin having a diameter of 1 to 2 mm.
3. Staining of Shigella: Same as mentioned under gram-positive bacteria staining.
4. Culture of Shigella: Selective media or differential media for shigella are DCA, XLD, CLED, MacConkey agar, and Hektoen enteric media. DCA and MacConkey agar media are the most commonly used culture media. The optimum temperature for inoculation of Shigella is 370C. After an overnight inoculation period, non-lactose fermenting colonies are isolated and identified.
5. Biochemical Characteristics of Shigella: Shigella gives a negative test for oxidase test, urease test, citrate test, H2S test, lactose test, sucrose test, and Voges-Proskaeur test. Shigella gives a positive glucose test. Shigella is a non-motile bacteria.
Biochemical tests Reactions
1. Citrate test: Negative
2. H2S test: Negative
3. Indole test: Different strains have a different result
4. Oxidase test: Negative
5. Urease test: Negative
6. VP: Negative
7. Urease: Negative
8. Gelatin: Negative
9. PPA: Negative
10 Nitrate test: Positive
11. MR: Positive
12. TSI
13. Base: Acid, Slant: Alkaline
Sugar Fermentation Carbohydrate Reactions
1. Glucose: Positive, Generate acid
2. Lactose: Negative
3. Sucrose: Negative
4. Maltose: Negative
5. Xylos: Negative
6. Mannitol: Negative
6. Antibiotics related to Shigella: Shigella is sensitive to Ampicillin, chloramphenicol, cephalosporins, and aminoglycosides.
7. Distribution of Shigella: Human intestine as parasite.