Microbiological Identification of Medically Important Staphylococci PDF

Microbiological identification of Medically important staphylococci by Heba Rashed Introduction to Staphylococci Ubiquitous Presence Opportunistic Pathogens Staphylococci are Gram-positive While many Staphylococcus bacteria that are commonly found species are harmless...

Microbiological identification of Medically important staphylococci by Heba Rashed Introduction to Staphylococci Ubiquitous Presence Opportunistic Pathogens Staphylococci are Gram-positive While many Staphylococcus bacteria that are commonly found species are harmless on the skin and mucous commensals, some can cause membranes of humans and serious infections in animals. immunocompromised individuals. Medically Significant Species Antibiotic Resistance Concerns The most clinically relevant Many Staphylococcus strains Staphylococcus species are have developed resistance to Staphylococcus aureus and common antibiotics, making coagulase-negative treatment of infections Staphylococci (CoNS). challenging. Staphylococcus aureus Staphylococcus aureus Morphology: are Gram-positive cocci arranged in clusters. Cultural characters: Staphylococci are facultative anaerobes that grow by aerobic respiration or microaerophilic at 37C. The colonies are round , smooth, raised , grey to golden yellow Asymptomatic Colonization of S. aureus Some are members of normal flora of skin and mucous membrane of humans as 40-50% of population are nasal carriers. Can colonize moist skin folds, oropharynx, GI tract, urogenital tract 15-20% of healthy adults are persistent nasopharyngeal carriers Higher number of carriers amongst hospital staff Virulence factors Coagulase enzyme; is an enzyme like protein that clots citrated plasma Exfoliative toxin Enterotoxins causing food poisoning DNase Catalase Haemolysins, they are responsible for β haemolysis on blood agar  S. aureus Reservoir Present in most environments frequented by humans Readily isolated from fomites Carriage rate for healthy adults is 20-60% Carriage is mostly in anterior nares, skin, nasopharynx, intestine Sample Collection and Processing Specimen Collection Collect appropriate clinical samples, such as nasal swabs, skin lesions, or blood, following proper sterile techniques. Initial Processing Transport the samples to the laboratory promptly and inoculate them onto appropriate culture media. Incubation Incubate the inoculated media at 35-37°C for 18-24 hours to allow for bacterial growth. Examination Examine the culture plates for the presence of colonies characteristic of staphylococci, such as golden-yellow or white, smooth, and convex. laboratory identification of Staphylococcus aureus Specimen : according to site of infection Direct smear stained with Gram stain shows Gram-positive cocci arranged in clusters among the pus cells. laboratory identification of Staphylococcus aureus Culture the samples on 1. Blood agar to test hemolytic reactions 2. Nutrient agar shows endopigment production Golden yellow pigment ( Staph. aureus ) Lemon yellow pigment ( Staph. saprophyticus) White pigment ( Staph. epidermidis) The Gram Stain: Identifying Staphylococci Prepare the Smear 1 Carefully transfer a small sample of the bacterial colony onto a clean microscope slide. 2 Crystal Violet Staining Flood the smear with crystal violet Gram Decolorization dye and allow it to sit for 1 minute. 3 Rinse the slide with a gentle stream of Gram's iodine solution to Counterstain with Safranin decolorize the sample. 4 Apply safranin dye for 30 seconds to stain any Gram-negative bacteria present.  Gram Stain Appearance Staphylococci are Gram-positive cocci that appear as clusters resembling grapes when viewed under a microscope after Gram staining. The cells typically stain a deep purple color, indicating their Gram- positive nature. The Gram stain is a crucial first step in the identification of Staphylococcus species, as it helps differentiate them from Gram-negative bacteria and other Gram-positive cocci. Culture media and growth characteristics Primary Isolation Growth Conditions Pigment Production Hemolysis Staphylococci are Staphylococci are Many Staphylococcus On blood agar, some typically cultured on facultative anaerobes, species produce a Staphylococcus species nutrient-rich media like growing best in the golden-yellow pigment exhibit alpha (partial) blood agar or mannitol presence of oxygen but called carotenoid, which or beta (complete) salt agar. They form also able to grow can help differentiate hemolysis, which can characteristic round, anaerobically. They them from other provide additional clues raised, and golden- prefer a temperature genera. to identification. yellow colonies. of 35-37°C and a neutral pH. Biochemical tests It produces complete haemolysis on blood agar. The organism produces catalase positive and coagulase positive. It ferments mannitol on mannitol salt agar and is differentiating value from other species. Catalase Test Microscopic Examination Observe the morphology of the bacterial cells under a microscope to confirm they are Gram-positive cocci arranged in clusters. Sample Preparation Obtain a fresh bacterial colony and suspend it in a drop of hydrogen peroxide solution on a clean slide. Catalase Reaction Observe for immediate production of bubbles, indicating a positive catalase test and confirming the bacteria as Staphylococci. 20 Key reactions: catalase & coagulase S. aureus Flashcard Catalase test : it is used to differentiate between streptococci (catalase-negative) from staphylococci (catalase positive). of catalase The test is performed by 3% or 10 % hydrogen peroxide to a test colony on an agar plate or slant Catalase-positive cultures produce bubbles Coagulase Test Principle Procedure A small amount of the The coagulase test detects bacterial culture is mixed the presence of the enzyme with rabbit plasma, and the coagulase, which is produced mixture is incubated. by pathogenic staphylococci Coagulase-positive and can cause blood clotting. staphylococci will cause the plasma to clot. Interpretation A positive coagulase test indicates the presence of Staphylococcus aureus, which is a major human pathogen. Negative results suggest the presence of less virulent staphylococcal species. Tube coagulase test Aim of experiment : this test for the bacteria's ability to clot blood plasma using the enzyme coagulase ( convert fibrinogen to fibrin) Name of reagent: rabbit or human plasma Principle of coagulase test: Coagulase is an enzyme-like protein and causes plasma to clot by converting fibrinogen to fibrin. Staphylococcus aureus produces two forms of coagulase: bound and free. Membrane bound coagulase (clumping factor) can be detected by clumping of bacterial cells in the slide coagulase test Extracellular free coagulase can be detected by change the plasma into clot in the tube coagulase test. Slide coagulase test 1. Place a drop of saline on each end of a slide 2. 2. With the bacterial loop emulsify a portion of the isolated colony in each drop to make two thick suspensions. 3. 3. Add a drop of undiluted human or rabbit plasma to one drop of suspension and mix gently. 4. 4. Observe the clumping of the organisms within 10 seconds. 5. 5. No plasma is added to the second suspension to differentiate any granular appearance of the organism from true coagulase clumping DNase Test Purpose Procedure Interpretation The DNase test is used to A bacterial colony is A positive DNase test, detect the production of inoculated onto a DNase along with other the enzyme test agar plate and biochemical tests, helps deoxyribonuclease incubated. The presence differentiate (DNase) by bacteria, of a clear zone around the Staphylococcus from which is an important colony indicates a positive other Gram-positive cocci characteristic for the result, which is and is an important step in identification of characteristic of the identification of Staphylococcus species. Staphylococcus species. medically relevant Staphylococcus species.  Mannitol fermentation Mannitol Fermentation Positive Reaction Testing Procedure Staphylococci can be Pathogenic Staphylococcus The mannitol fermentation differentiated based on aureus can ferment test is routinely performed their ability to ferment mannitol, turning the media in the clinical microbiology mannitol, an important yellow, while coagulase- lab to identify and carbohydrate test. negative staphylococci do differentiate not. Staphylococcus species. Flashcard of Mannitol salt agar ▪ Mannitol salt agar is selective and differential medium for Staphylococci group Selective by 7.5% sodium chloride (NaCl) ▪ Differential by mannitol sugar ▪ pH indicator is phenol red at pH 7.3 ▪ Staphylococcus aureus ferment mannitol produces yellow ( pH acidic) ▪ Coagulase negative staph. does not ferment mannitol and the color remain ( red) Antibiotic Susceptibility Testing Disk Diffusion Minimum Inhibitory Concentration (MIC) This is a standard method that measures the zone of inhibition The MIC is the lowest concentration of an antibiotic that around antibiotic disks to determine susceptibility. prevents visible bacterial growth, providing a more precise measurement. Automated Systems Molecular Techniques Automated platforms can rapidly identify bacteria and test their Genetic tests can detect antibiotic resistance genes, guiding susceptibility to a panel of antibiotics. targeted treatment and tracking emerging resistance patterns. S. aureus diseases Ranges from localized to systemic localized -abscess, folliculitis, furuncle, carbuncle, impetigo systemic – osteomyelitis, bacteremia toxigenic disease – food intoxication, scalded skin syndrome, toxic shock syndrome Case studies and clinical relevance Osteomyelitis Case Surgical Site Infection MRSA Bacteremia A 45-year-old patient presented with A 60-year-old patient developed a A 75-year-old patient with multiple persistent joint pain and was Staphylococcus epidermidis surgical comorbidities acquired a methicillin- diagnosed with a Staphylococcus site infection following an orthopedic resistant Staphylococcus aureus aureus osteomyelitis infection after procedure, requiring intensive (MRSA) bloodstream infection during laboratory testing. antibiotic treatment. their hospital stay, leading to sepsis. Novobiocin disc test Differentiation between Staph epidermidis and saprophyticus By Novobiocin disc test Micrococcus Common saprophyte in air, water and soil. Gram + ve cocci found in tetrads than clusters. Catalase + ve & coagulase – ve. May give lemon yellow colonies. Acts on glucose oxidatively. Micrococcus is similar to Staph. epidermidis. It can be differentiate between Staph. epidermidis and Micrococcus by Bacitracin test (0.04- 0.05 IU) Micrococcus is sensitive. Staph epidermidis is resistant Micrococcus colonies on nutrient agar Methicillin resistant staph aureus ( MRSA) Nearly 95% of staphylococcus aureus strains are resistant to penicillin. Methicillin-resistant staphylococcus aureus ( MRSA) it is more serious type of resistance which cannot be treated with any of beta lactam antibiotics MRSA are resistant to most antibiotics Drug of choice is vancomycin Hospital-associated methicillin-resistant Staphylococcus aureus (HA-MRSA) and community-associated MRSA (CA-MRSA) are: Resistance for both strains is conferred by the mecA gene. HA strains tend to demonstrate resistance to more drug classes than CA strains. Which of the following statements about Staphylococci is incorrect? A. They are all pathogenic. B. They are Gram-positive bacteria. C. They can be facultative anaerobes. D. They can colonize mucous membranes. Which of the following statements about Staphylococci is incorrect? A. They are all pathogenic. B. They are Gram-positive bacteria. C. They can be facultative anaerobes. D. They can colonize mucous membranes. ANSWER: A Which biochemical test can help differentiate species of Staphylococci? A. Catalase test B. Sugar fermentation C. Coagulation time D. Oxidase test Which biochemical test can help differentiate species of Staphylococci? A. Catalase test B. Sugar fermentation C. Coagulation time D. Oxidase test ANSWER: A Staphylococci cannot cause infections in immunocompromised individuals. A. True B. False Staphylococci cannot cause infections in immunocompromised individuals. A. True B. False ANSWER: B Staphylococcus epidermidis typically produces a golden yellow pigment. A. True B. False Staphylococcus epidermidis typically produces a golden yellow pigment. A. True B. False ANSWER: B Staphylococcus aureus can produce enzymes such as ______, which is responsible for clotting citrated plasma. Staphylococcus aureus can produce enzymes such as ______, which is responsible for clotting citrated plasma. ANSWER: coagulase Approximately ______% of the healthy adult population are persistent nasopharyngeal carriers of S. aureus. Approximately ______% of the healthy adult population are persistent nasopharyngeal carriers of S. aureus. ANSWER: 15-20 Match the following Staphylococcus species with their characteristic pigments: Staphylococcus hominis | Lemon yellow Staphylococcus epidermidis | Colorless Staphylococcus saprophyticus | Golden yellow Staphylococcus aureus | White Staphylococcus aureus = Golden yellow Staphylococcus epidermidis = White Staphylococcus saprophyticus = Lemon yellow Staphylococcus hominis = Colorless