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 species are harmless found on...

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 species are harmless found on the skin and mucous commensals, some can cause membranes of humans and serious infections in animals. immunocompromised individuals. Medically Significant Antibiotic Resistance Species 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 Staphylococci are Gram-positive spherical bacteria arranged in grapes. They produce endopigment that vary from white to yellow. Staphylococcus aureus (golden yellow) and Staphylococcus epidermidis (white) and staphylococcus saprophyticus ( lemon 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 adlts are persistent nasopharyngeal carriers Higher number of carriers amongst hospital staff  Spherical cells arranged in irregular clusters Gram positive General characteristics of Catalase- positive the Staphylococci Common inhabitant of the skin & mucous membranes 31 species Virulence factors 1. Coagulase enzyme; is an enzyme like protein that clots citrated plasma 2. Exfoliative toxin 3. Enterotoxins causing food poisoning 4. DNase 5. Catalase 6. Haemolysins, they are responsible for β haemolysis on blood agar Enzymes of S. aureus Coagulase – coagulates plasma and blood; produced by 97% of human isolates differentiates S.aureus from other staphylococci Hyaluronidase Staphylokinase: activates plasminogen to form plasmin, which digest fibrin clots DNAse Lipases Penicillinase Toxins of S. aureus hemolysins – lyse RBCs; α-toxin , β-toxin and δ-toxin Panton-Valentine Leukocidin (PVL) enterotoxins exfoliative toxin toxic shock syndrome toxin  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 Process 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. 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 dye and allow it to sit for 1 minute. Gram Decolorization 3 Rinse the slide with a gentle stream of Gram's iodine solution to decolorize the sample. 4 Counterstain with Safranin 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 typically Staphylococci are facultative Many Staphylococcus species On blood agar, some cultured on nutrient-rich media anaerobes, growing best in the produce a golden-yellow Staphylococcus species exhibit like blood agar or mannitol salt presence of oxygen but also able pigment called carotenoid, alpha (partial) or beta agar. They form characteristic to grow anaerobically. They which can help differentiate (complete) hemolysis, which can round, raised, and golden- prefer a temperature of 35- them from other genera. provide additional clues to yellow colonies. 37°C and a neutral identification. pH.  Morphology and characteristics Cell Morphology Colonial Appearance Ultrastructure Staphylococci are gram-positive On culture media, Staphylococcus At the ultrastructural level, cocci that typically arrange in grape- colonies are circular, raised, and Staphylococci have a thick like clusters under the microscope. often have a golden-yellow pigment. peptidoglycan cell wall and divide in multiple planes. Differential Biochemical Tests Culture Growth Biochemical Reactions Observing the growth Performing specific biochemical tests, such as characteristics of catalase, coagulase, and DNase, can provide staphylococci on different key identifying information. culture media can help differentiate species. Microscopic Morphology Antibiotic Susceptibility Analyzing the cellular arrangement and Determining the antimicrobial resistance Gram stain characteristics under the profile can help distinguish between clinically microscope is crucial for identification. relevant staphylococcal species. 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. Key reactions: catalase & coagulase S. aureus Flashca Catalase test : it is used to differentiate between streptococci (catalase-negative) from staphylococci (catalase positive). rd of catalas The test is performed by 3% or 10 % hydrogen peroxide to a colonyon an agar plate or slant e test Catalase-positive cultures produce bubbles Coagulase Test Principle Procedure A small amount of the bacterial culture The coagulase test detects the presence is mixed with rabbit plasma, and the of the enzyme coagulase, which is mixture is incubated. Coagulase- produced by pathogenic staphylococci positive staphylococci will cause the and can cause blood clotting. 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 A bacterial colony is A positive DNase test, to detect the production inoculated onto a DNase along with other of the enzyme test agar plate and biochemical tests, helps deoxyribonuclease incubated. The presence differentiate (DNase) by bacteria, of a clear zone around Staphylococcus from which is an important the colony indicates a other Gram-positive characteristic for the positive result, which is cocci and is an important identification of characteristic of step in the identification Staphylococcus Staphylococcus of medically relevant species. species. Staphylococcus species.  Mannitol fermentation Mannitol Fermentation Positive Reaction Testing Procedure Staphylococci can be differentiated Pathogenic Staphylococcus aureus can The mannitol fermentation test is based on their ability to ferment ferment mannitol, turning the media routinely performed in the clinical mannitol, an important carbohydrate yellow, while coagulase-negative microbiology lab to identify and test. staphylococci do not. differentiate Staphylococcus species. Flashcard of Mannitol salt agar  Mannitol salt agar is selective and differential medium for Staphylococci group Selective by 7.5% sodium cloride (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 The MIC is the lowest concentration of an antibiotic that inhibition around antibiotic disks to determine prevents visible bacterial growth, providing a more precise susceptibility. measurement. Automated Systems Molecular Techniques Automated platforms can rapidly identify bacteria and test Genetic tests can detect antibiotic resistance genes, their susceptibility to a panel of antibiotics. guiding 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 resistant Staphylococcus aureus aureus osteomyelitis infection after orthopedic procedure, requiring (MRSA) bloodstream infection during laboratory testing. intensive antibiotic treatment. their hospital stay, leading to sepsis.  Other Staphylococci (Coagulase- negative) All may cause wound infections e.g. S. epidermidis – lives on skin & mucous membranes; etiology of endocarditis, bacteremia on indwelling catherters), UTI Novobiocin disc test Differentiation between Staph epidermidis and saprophyticus By Novobiocin disc test Novobiocin Susceptibility 1 Staphylococcus 2 Novobiocin Identification Sensitivity Novobiocin susceptibility S. aureus is typically is a key test for susceptible to novobiocin, differentiating between while S. saprophyticus is Staphylococcus species. resistant. 3 Diagnostic 4 Test Procedure Significance Novobiocin disks are This test helps distinguish placed on an inoculated between these two agar plate and the zone medically important of inhibition is measured. Staphylococcus species. 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 Clinical concerns 95% have penicillinase & are resistant to penicillin & ampicillin MRSA – methicillin- resistant S. aureus – carry multiple resistance Abscesses have to be surgically drained Systemic infections require intensive lengthy therapy Methicillin resistant staph aureus ( MR SA) Nearly 95% of staphylococcus aureus strains are resistant to penicillin. Methicillin-resistant staphylococcus aureus ( MR SA) it is more serious type of resistance which cannot be treated with any of beta lactam antibiotics MR SA are resistant to most antibiotics Drug of choice is vancomycin Hospital-associated methicillin-resistant Staphylococcus aureus (HA-MR SA) and community-associated MR SA (CA-MR SA) are: Resistance for both strains is conferred by the mecA gene. HA strains tend to demonstrate resistance to more drug classes than CA strains. Importance in Healthcare Settings 1 Infection Prevention 2 Diagnostic Accuracy Identifying and containing Reliable microbiological staphylococcal infections is identification of Staphylococcus crucial to prevent the spread of species is essential for accurate antibiotic-resistant strains in diagnosis and targeted antibiotic hospitals and other healthcare treatment of infections. facilities. 3 Outbreak Management 4 Antibiotic Stewardship Rapid detection and Identifying Staphylococcus characterization of susceptibility patterns guides Staphylococcus outbreaks appropriate antibiotic selection, allows healthcare teams to supporting antimicrobial implement effective control stewardship programs to measures and prevent further combat drug resistance. transmission.