Medically Important Bacteria Gram Positive Cocci

Group B b-Hemolytic Streptococcus (Streptococcus agalactiae) Has been known to cause mastitis in cattle Colonize the of urogenital tract pregnant women Cause invasive diseases in newborns Early-onset infection Late-onset disease

Streptococcus agalactiae: Invasive Infections Early-onset infection Occurs in neonates who are less than 7 days old neonates Vertical transmission of the organism from the mother Manifests in the form of pneumonia or meningitis with bacteremia Associated with a high mortality rate

Streptococcus agalactiae: Invasive Infections Late-onset infection Occurs between 1 week and 3 months after birth Usually occurs in the meningitis form Mortality rate is not as high as early-onset In adults Occurs in immunosuppressed patients or those with underlying diseases Often found in a previously healthy adult who just experienced childbirth

Laboratory Diagnosis: Group B b-Hemolytic Streptococcus Presumptive identification tests Bile-esculin-hydrolysis–negative Does not grow in 6.5% NaCl CAMP-test–positive Hippurase positive S. agalactiae shows the arrow-shaped hemolysis near the staphylococcus streak, showing a positive test for CAMP factor

Bile Esculin test Bile-esculin test is based on the ability of certain bacteria to hydrolyze esculin in the presence of bile (4% bile salts or 40% bile). Bacteria that are bile-esculin positive are, first of all, able to grow in the presence of bile salts. Hydrolysis of the esculin in the medium results in the formation of glucose and a compound called esculetin. Esculetin, in turn, reacts with ferric ions (supplied by the inorganic medium component ferric citrate) to form a black diffusible complex.

Hippurate test Hippurate hydrolysis test is used to detect the ability of bacteria to hydrolyse hippurate into glycine and benzoic acid by action of hippuricase enzyme present in bacteria. an oxidizing agent ninhydrin is used as an indicator. Ninhydrin reacts with glycine to form a deep blue or purple color (purple).

Mode of control Penicillin G is the drug of choice A combination from penicillin and aminoglycoside is used in patients with serious infection. Vancomycin is used for patients allergic to penicillin.

Identification Schema Schema to differentiate Group A and B from other b-hemolytic streptococci

Streptococcus Group D and Enterococcus Species Members of the gut flora Associated infections Bacteremia Urinary tract infections Wound infections Endocarditis

Group D Streptococcus Enterococcus – 2 imp. species E. fecalis 1 Enterococcus – 2 imp. species E. fecalis E. faecium Normal flora in GIT, lower genital tract Nosocomial / opportunistic pathogen Resistance to cephalosporins, even vancomycin

Laboratory Diagnosis: Streptococcus Group D and Enterococcus Species Microscopic morphology Cells tend to elongate Colony morphology Most are non-hemolytic, although some may show a- or, rarely, b-hemolysis Possess Group D antigen

Laboratory Diagnosis: Streptococcus Group D and Enterococcus Species Identification tests Catalase: may produce a weak catalase reaction Hydrolyze bile esculin Differentiate Group D from Enterococcus sp. with 6.5% NaCl Penicillin resistance

Identification Schema Schema to differentiate Enterococcus and Group D streptococci from other nonhemolytic streptococci

Other Streptococcal Species Viridans group Members of the normal oral and nasopharyngeal flora Includes those that lack the Lancefield group antigen Most are  hemolytic but also includes nonhemolytic species The most common cause of subacute bacterial endocarditis (SBE)

Streptococcus pneumoniae General characteristics Inhabits the nasopharyngeal areas of healthy individuals Typical opportunist Possess C substance Virulence factors Polysaccharide capsule Clinical infections Pneumonia - meningitis Bacteremia - sinusitis/otitis media

Laboratory Diagnosis: Streptococcus pneumoniae Microscopic morphology Gram-positive cocci in pairs; lancet-shaped

Laboratory Diagnosis: Streptococcus pneumoniae Colony morphology Smooth, glistening, wet-looking, mucoid a-Hemolytic CO2enhances growth

Laboratory Diagnosis: Streptococcus pneumoniae Identification Catalase negative Optochin-susceptibility-test–susceptible Bile-solubility-test–positive

Identification Schema Schema to differentiate S. pneumoniae from other a-hemolytic streptococci

Differentiation between -hemolytic streptococci The following definitive tests used to differentiate between S. pneumoniae & viridans streptococci Optochin Test Bile Solubility Test Inulin Fermentation

Optochin Susceptibility Test Principle: Optochin (OP) test is presumptive test that is used to identify S. pneumoniae S. pneumoniae is inhibited by Optochin reagent (<5 µg/ml) giving a inhibition zone ≥14 mm in diameter. Procedure: BAP inoculated with organism to be tested OP disk is placed on the center of inoculated BAP After incubation at 37oC for 18 hrs, accurately measure the diameter of the inhibition zone by the ruler ≥14 mm zone of inhibition around the disk is considered as positive and ≤13 mm is considered negative S. pneumoniae is positive (S) while S. viridans is negative (R)

Optochin Susceptibility Test Optochin resistant S. viridans Optochin susceptible S. pneumoniae

Bile Solubility test Principle: Procedure: S. pneumoniae produce a self-lysing enzyme to inhibit the growth The presence of bile salt accelerate this process Procedure: Add ten parts (10 ml) of the broth culture of the organism to be tested to one part (1 ml) of 2% Na deoxycholate (bile) into the test tube Negative control is made by adding saline instead of bile to the culture Incubate at 37oC for 15 min Record the result after 15 min

Bile Solubility test Results: Positive test appears as clearing in the presence of bile while negative test appears as turbid S. pneumoniae soluble in bile whereas S. viridans insoluble

Differentiation between -hemolytic streptococci CAMP test Bacitracin sensitivity Hemolysis Negative Susceptible  S. pyogenes Positive Resistant S. agalactiae Differentiation between -hemolytic streptococci Inulin Fermentation Bile solubility Optochin sensitivity Hemolysis Not ferment Soluble Sensitive (≥ 14 mm)  S. pneumoniae Ferment Insoluble Resistant (≤13 mm) Viridans strep

Points to Remember General characteristics and hemolytic patterns of streptococcal and enterococcal species Infections produced by pathogenic species Microscopic and colony morphology Tests used to identify these species Emergence of resistant strains