1. Diagnostic microbiology lecture: 8 THE GRAM POSITIVE COCCI Abed ElKader Elottol MSc. Microbiology 2010 1

2. 2

3. 3

4. Species 33 species are known. Three are medically important: 1. Staphylococcus aureus Most important pathogen 2. Staphylococcus epidermidis May cause endocarditis 3. Staphylococcus saprophyticus May cause cystitis. 4

5. General Characteristics 1. Cocci arranged in grape-like clusters 2. Strongly gram-positive 3. Ferments many carbohydrates with the production of lactic acid but no gas 4. Non-motile 5. Non-spore forming 5

6. Staphylococcus aureus Aureus: golden color (golden color colonies on blood agar) Diseases caused by the organism: 1. Toxic shock syndrome 2. Furuncles (abscess) 2. Septicemia 4. Impetigo 5. Meningitis 6. Pneumonia 7. Food poisoning 8. Pyoderma 6

7. 7

8. Cultural and Morphological Characteristics 1.Media for Primary Isolation: S. aureus grow well in most routine media e.g,Blood Agar, Nutrient Agar. 2. Media for Selective Isolation: Mannitol Salt Agar (MSA), is an excellent medium which contains 7.5% sodium chloride which is considered as a high percentage and inhibitory to most medically important bacteria. In addition it contains mannitol as the only carbon source and a pH indicator to detect mannitol fermentation by S. aureus. 3. Incubation: After streaking the specimen on one of the common media, incubate the plates at 35-37 oC for 24 hours. 8

9. Colony Morphology On blood agar plates: colonies are 2-4 mm in diameter, rounded and slightly elevated. Most pathogenic strains produces a zone of β-hemolysis. Another distinguishing character is the production of a golden yellow pigment. On Mannitol Salt Agar: The colonies are surrounded by a yellow zone indicative of acid production resulting from the fermentation of Mannitol. 9

10. Gram Stained Smears: Gram-positive cocci arranged in clusters. Single cells, diplococci, and short chains may also appear. It is usually simple to identify the morphology in stained film from sputum or pus but one can be certain by performing simple biochemical tests for the isolate e.g., catalase test to differentiate it from Streptococci and Coagulase or DNase to differentiate it from non-pathogenic staphylococci.. 10

11. 11

12. Biochemical Characteristics 1. Catalase positive 2. Mannitol fermenter 3. Grow well in 7.5% NaCl 4. Coagulase positive 5. DNase positive 6. Glucose fermenter. 12

13. 13

14. Identification: 1. Based on gram-staining 2. To differentiate it from other gram-positive cocci (Streptococci) perform catalase test 3. To differentiate it from other non-pathogenic staphylococci a group of biochemical tests are performed. a. Coagulase test b. Mannitol fermentation c. Growth in 7.5% NaCl d. Glucose OF e. DNase 14

15. PHAGE TYPING A Staphylococcus aureus-specific phage is added to a plate that is inoculated with S.aureus. The plates are incubated at 37 oC for 24 hours. Positive identification: Formation of plaques Negative results: S. aureus grow over the whole area. 15

16. Sensitivity testing and treatment S. aureus is a frequent hospital pathogen and it has the ability to develop resistance to the commonly known antibiotics. For this reason sensitivity testing must be performed on all isolates. Penicillin G and its derivatives (ampicillin, amoxycillin, cloxacillin, methicillin), ofloxacillin and cephalosporins are usually effective against S. aureus. 16

17. ANTIBIOTICS RESISTANCE Historical aspect 1940s : all S. aureus were sensitive to penicillin Shortly after use : penicillin resistant strains appeared which produced betalactamase rapidly spread In late 1950s : beta-lactamase - resistant penicillin (methicillin) (not degraded by) In 1961 methicillin-resistant S. aureus (MRSA) was discovered (presently a major problem) 17

18. MRSA Low carriage rate in community High in tertiary care hospitals Mode of Transmission Fomites Direct from hospital staff or attendants : contaminated hands 18

19. MRSA causes a variety of disseminated, lethal infections in humans. Has the ability to easily transfer resistant genes to other species directly and indirectly. Overuse of antibiotics imposes selective pressures which mediates the acquisition of resistance. Most major organs fail with disseminated MRSA. 19

20. PREVENTION OF STAPH INFECTIONS Control of Carrier and reinfection Wash clothes in hot water (>70oC) Use antiseptic soap (Dettol soap) Antimicrobial nasal cream (Gentamicin, Mupirocin) Oral antibiotics that are concentrated in nasal secretions (ciprofloxacin and rifampicin) Chemoprophylaxis :Antibiotics before and at time of surgical operation 20

21. Negative Oxidase test

22. 22 COAGULASE-NEGATIVE STAPHYLOCOCCI (CNS )

23. 23 Normal flora in ° Skin ° Anterior nose ° External ear canal Cell wall contains teichoic acid (glycerol type) White, non-haemolytic colonies on blood agar Sensitive to novobiocin; (S. saprophyticus is resistant)

24. 24 DISEASES BY S. EPIDERMIDIS Most infections are hospital acquired Opportunistic pathogen in immuno-suppressed Strongly associated with presence of foreign bodies ° Prosthetic heart valves (endocarditis) ° IV catheters (bacteremia) ° Urinary catheter (UTI in elderly) ° CSF shunts (meningitis) ° Peritoneal dialysis catheter (peritonitis)

25. 25 Staphylococus saprophyticus

26. 26 Saprophytic in life. Resistant to novobiocin. Most infections are community-acquired. ° Primary UTI in 10-20% of young adult women. hormonal factors may be involved. Resistant to antibiotics – penicillins & cephalosporins

27. 27 The End