Clinical Bacteriology - Lecture - Staphylococcus PDF

CLINICAL BACTERIOLOGY - LECTURE MIDTERMS TOPIC 1. MEDICALLY IMPORTANT GRAM POSITIVE COCCI Lecturer/s: Mr. Jason “Ab” Chua, RMT, MSMT September 16, 2023 FULL TRANSES MASTERLIST: https://bit.ly/masterli_st...

CLINICAL BACTERIOLOGY - LECTURE MIDTERMS TOPIC 1. MEDICALLY IMPORTANT GRAM POSITIVE COCCI Lecturer/s: Mr. Jason “Ab” Chua, RMT, MSMT September 16, 2023 FULL TRANSES MASTERLIST: https://bit.ly/masterli_st GRAM POSITIVE COCCI Ribosomal RNA & DNA Hybridization Analysis – it ↑ peptidoglycan; ↓ level of lipid (cell wall) was found out that 2 families are present ○ Alcohols and other solvents cannot ○ Recent Edition: (2 families): penetrate the gram (+) cell wall. Instead, it Family Staphylococcaceae causes the cell wall to shrink so it locks in Family Micrococcaceae the primary stain (crystal violet) Family Staphylococcaceae Natural inhabitants of skin & mucous membrane Includes: (GSM-S) (normal microbiota) Genus Staphylococcus Can be ubiquitous in the environment ○ Major human pathogen (SIZE: 1-2 ○ Can be found ANYWHERE micrometers) Infection → transmitted via direct contact Genus Gemella ○ e.g. infected individuals; contaminated Genus Macrococcus objects ○ Similar to staphylococcus but produces The infection will lead to the accumulation of slightly larger colonies (1-2.5 micrometers) neutrophils, bacterial cells and fluids at the site = ○ Relatively non-pathogenic PYOGENIC INFECTION (Presence of PUS) due to Genus Salinicoccus Staphylococcal infection Family Micrococcaceae Includes: LIST OF GRAM POSITIVE COCCI Genus Micrococcus Staphylococcus Stomatococcus ○ Found in: tetrads, pairs, or clusters (SIZE: 0.5 - 1.5 micrometers) Rothia Leuconostoc ○ Rarely infectious Genus Arthrobacteria Planococcus Streptococcus Genus Kocuria Micrococcus Alloiococcus Stomatococcus mucilaginosus Only member of the Genus Stomatococcus Gemella Pediococcus Reclassified under the Genus Rothia ○ Rothia mucilaginosus Macrococcus Lactococcus New name ; considered as a normal microbiota of the human Salinicoccus Aerococcus respiratory tract Among ALL these organisms, only TWO ARE Cannot grow in the presence of 5% NaCl IMPORTANT WEAKLY CATALASE (+), COAGULASE (-) and has a ○ Staphylococcus and Streptococcus CAPSULE ○ Staphylococcus and Micrococcus are the ○ Mucoid-slimy characteristic same in terms of Shape, appearance, and Emerging pathogen in IMMUNOSUPPRESSED both of them are CATALASE POSITIVE. PATIENTS ○ We should be able to differentiate if ○ E.g. cancer patients, transplant, burn Staphylococcus/Micrococcus are present in patients, AIDS patients Micrococcus vs. Staphylococcus ○ Micrococcus rarely infects patient; Staphylococcus commonly causes human Micrococcus Staphylococcus infection Growth Characteristics ○ Streptococcus is CATALASE NEGATIVE Reclassification of Gram Positive Cocci Oxygen Facultative In 1996: Bergey’s Manual of Systematic Obligate aerobe Requirement anaerobe Bacteriology Aerobic Growth ○ Family Micrococcaceae includes 4 genera: Micrococcus + + Staphylococcus Anaerobic Planococcus Growth - + Stomatococcus Made by Miguel Astronomo, Daniel Budo Page 1 of 9  Staphylococcus aureus Antibiotic Susceptibility **BE FAMILIAR WITH THIS PART OF THE TABLE.** GENERAL CHARACTERISTICS Bacitracin (0.04 Susceptible Resistant unit) Furazolidone Resistant Susceptible (100 ug/mL) Lysostaphin (200 Resistant Susceptible ug/mL) Carbohydrate Utilization (OF Oxidative Fermentative Medium) Open Tube Yellow (+) Yellow (+) Closed Tube Green (-) Yellow (+) 10 to 30% carriers Transmission Modified ○ Direct contact Oxidase (Microdase) test + - ○ Fomites ○ Poor hygiene Growth on Infections Furoxone-Tween 80-oil red O agar + - ○ virulence of strain ○ size of inoculum Staphylococcus ○ immune status Significant human species: Staphylococcus aureus 1. S. aureus VIRULENCE FACTORS 2. S. epidermidis 3. S. saprophyticus 4. S. hominis 5. S. hemolyticus 6. S. warneri Common isolates: S. aureus, S. epidermidis, S. saprophyticus Staphylococcus: General Characteristics Clinically S. aureus Significant: S. epidermidis S. saprophyticus Catalase (+) Gram (+) cocci Nonmotile & Non-spore forming ○ They lack flagella that is supposed to be Surface structures used in locomotion Enzymes ○ Cannot produce spores (Only genus Bacillus Toxins and Clostridium) Staphylococcus aureus Facultative anaerobes SURFACE STRUCTURES Modified oxidase (-) Reduces nitrates to nitrites 1. Protein A Grows in 7.5% to 10% NaCl (Mannitol Salt Agar) a. binds to Fc region of IgG Lysostaphin sensitive - they are resistant to them b. Affects the immediate and Bacitracin resistant delayed hypersensitivity reactions → Protein A will now bind to the antibody, instead of the antibody Fc region → There will be no opsonization, phagocytic activity, and activation of the complement S. aureus will not be killed in this process and immune system / cells will not function well. Made by Miguel Astronomo, Daniel Budo Page 2 of 9  2. Capsular polysaccharide Staphylokinase a. Anti-phagocytic b. Enhance binding to host cells & prosthetics DNAse Breaks down DNA in the body → The capsule inhibits phagocytic process 3. Peptidoglycan and Teichoic Acid Beta lactamase Also known as Penicillinase a. Chemoattractant for neutrophils; Activates Breaks down B-lactam rings in complement which these are found in i. Attracts neutrophils somewhere antibiotics, specifically in penicillin making penicillin in the body, instead of the ineffective for Staphylococcus neutrophils going directly at the aureus site of infection b. Elicits production of IL-1 & opsonic Abs by TOXINS monocytes c. mediates adhesion by binding to tissue Exfoliatin Cleave stratum granulosum of fibronectin the skin Excessive peeling of skin Staphylococcus aureus Associated with ENZYMES AND TOXINS Staphylococcal Scalded Skin ENZYMES Syndrome Catalase will break down Hydrogen Leukocidin Also known as peroxide into water and oxygen Panton-Valentine Leukocidin (Bubble formation) (PVL) Hydrogen peroxide is needed by Toxin responsible for the immune cells to function continuously puncturing properly – no H2O2 means no holes in the immune cells immune action – no rendering them dead and opsonization, etc. inhibit phagocytosis Bound bound directly to the fibrinogen Hemolysins Lyse RBCs and erythrocytes coagulase [protein] in which if the which are toxic to the immune (Clotting coagulase is already bound to, cells Factor) will convert Fibrinogen into FOUR TYPES Fibrin which will result in a clot. 1. Alpha A clot in the body is toxic to the 2. beta immune cells 3. gamma TWO TYPES OF COAGULASE 4. delta 1. Bound coagulase (Clotting Factor) Enterotoxins Seven heat stable proteins 2. Free coagulase (Tube 1. A Coagulase) 2. B a.Reacts with the plasma 3. C coagulating reaction factor 4. D (PCRF) in which the action is 5. E the same– Converts 6. G Fibrinogen to Fibrin 7. H Among the seven heat stable, only two Staphylokinase Enzyme that dissolves the clot. are very common that can cause food The clot becomes small poisoning ➔ A and B particles – these STILL become toxic for the immune Superantigens This is where Enterotoxins cells belong, as well as the Spreads infection throughout the Toxic-Shock Syndrome Toxin body Type 1 (TSST-1) ○ Between TSST-1 and Lipase Hydrolyze lipids in the skin / if TSST-2, only TSST-1 ever the lipids have been is superantigenic hydrolyzed, this manifests Responsible for making SKIN INFECTIONS caused by cytokines in the body – too the Staphylococcus much cytokines will let them ○ boils, carbuncles, accumulate, and increased furuncles cytokines will cause havoc Staphylococcus aureus Hyaluronidase Hydrolyzes hyaluronic acid – DISEASE PROCESS it is responsible for the spread SAU ➔ invades tissues and produce toxins infection Organism spread (site of carriage) ➔ site of infection SPREADING FACTORS Hyaluronidase (breaks on the skin) Made by Miguel Astronomo, Daniel Budo Page 3 of 9  Organism ➔ blood ➔ lungs, bones, liver, brain, or Empyema heart Staphylococcus aureus DISEASES TYPES OF STAPHYLOCOCCAL DISEASES Local abscesses Focal suppuration Diseases caused by toxin elaboration LOCAL ABSCESSES Folliculitis ➔ Presence of pus in the lungs Endocarditis Sepsis Impetigo/ ➔ Inflammation of hair follicles - where it is Pyoderma supposed to grow ➔ Back, arms, buttocks Furuncles (Boils) ➔ Presence of pus ➔ a.k.a Abscess ➔ Common in toddlers/infants that are Carbuncles yet to develop immunity EXOTOXIN ELABORATION Food poisoning ○ Enterotoxins A & B ○ Nausea, vomiting & abdominal pain manifests within 1 hr or so ➔ Collection of boils left untreated May lead to diarrhea ➔ Common at the region of the nape Scalded skin syndrome Stye ○ Epidermolytic toxin/ exfoliatin = Exfoliation as it cleaves Stratum granulosum FOCAL SUPPURATION Not one area in the body but affects adjacent area Osteomyelitis ○ ○ Can be alleviated Toxic Shock Syndrome ○ Multisystemic disease ○ TSST–1 producing strains of Staphylococcus ➔ Skin is affected, then the bone is aureus inflamed next TOXIC SHOCK SYNDROME ➔ Left untreated, will require amputation ➔ Common in patients with diabetes Pneumonia Meningitis Made by Miguel Astronomo, Daniel Budo Page 4 of 9  DNAse (-) Susceptible to Novobiocin ○ Most important test to determine whether it is epidermidis spp. or saprophyticus spp. Staphylococcus epidermidis DISEASE HOSPITAL ACQUIRED / NOSOCOMIALLY ACQUIRED ○ catheters ○ prosthetic heart valve implantation ○ prosthetic devices: CNS shunts (patients with hydrocephalus), intravascular catheters ○ immunosuppression Manifestations Staphyloccocus saprophyticus ○ Low blood pressure causing septic shock GENERAL CHARACTERISTICS Associated with tampons in females Contaminant ○ Blood is a source of food by the bacteria important cause of UTI among sexually active creating a biofilm allowing entrance into the females body Catheter-associated UTI Virulence Factor Ability to adhere to epithelial cells of the urinary tract - lining of genitalia - first to be seen in the urine[squamous epithelial cells] ○ Clear, transparent Catalase (+) Coagulase (-) DNAse (-) Non-hemolytic on Blood Agar Plate MSA fermentation variable (sometimes they cannot ferment Mannitol) Resistant to 5 ug NOVOBIOCIN ○ Zone of inhibition size: 6 mm to 12mm ; susceptible at least 16mm Upper pic: Desquamation of palms and soles MRSA - Methicillin Resistant Staphylococcus aureus Lower: Diffuse erythematous rash Strains of SAU that are resistant to beta lactams COAGULASE NEGATIVE Staphylococcus (CoNS) (penicillinase enzyme) Staphylococcus epidermidis Many SAU strains produce B-lactamase & are GENERAL CHARACTERISTICS penicillin-resistant Normal flora of skin & mucous membranes Oxacillin, cloxacillin, and methicillin G (+) cocci in clusters ○ DRUG OF CHOICE: METHICILLIN Virulence factor TWO TYPES OF METHICILLIN RESISTANT Staphylococcus Biofilm production aureus cell surface & extracellular compounds Significant nosocomial infection (HA-MRSA) promote adherence of bacteria to surfaces of CA-MRSA (Community-Associated) prosthetic devices. ○ Staphylcoccal cassette chromosome (SCC) ○ mecA gene Penicillin-binding protein 2A (PBP2a) LABORATORY DIAGNOSTIC PROCEDURES AND PRINCIPLES OF TESTS SPECIMEN COLLECTION & PROCESSING Specimens: Wound White and creamy non-hemolytic growth on BAP Blood (+) growth on CNA (Columbia Naladixic Acid Sputum Agar) Urine Can grow but lacks fermentation on MSA (Mannitol Salt Agar) Do direct gram stain from specimen Coagulase (-) Gram (+) cocci & neutrophils are noted Made by Miguel Astronomo, Daniel Budo Page 5 of 9  ○ If Gram (+) automatically perform catalase test Staphylococcus: Culture medium Chromogenic Agar (Hardy Chrome Staphylococcus aureus agar) SAU appears deep pink to fuchsia colonies S. epidermidis partially or completely inhibited Sheep's Blood Agar Plate S. saprophyticus appears turquoise colonies Medium-large in size, convex, creamy, dome shaped Staphylococcus aureus ○ Beta-hemolytic, some Staphylococcus spp. MACROSCOPIC CHARACTERISTICS have no hemolysis COLONIES Pigment: white to golden yellow Medium to large smooth, butyrous (butter–like), creamy Margin is entire (uniform shape/size) Columbia Colistin Nalidixic Acid Agar (CNA) Staphyloxanthin (yellow pigment) Selective medium for growth of G(+) bacteria Narrow zone of B-hemolysis (SBA) The medium is blood agar base with Antibiotics TEST FOR DIFFERENTIATION CATALASE TEST (Test for Differentiation) Differentiate Staphylococcus from Streptococcus Growth on MSA Selective & differential medium ↑ salt (7.5%) = selective Mannitol and phenol red COAGULASE TEST (Test for Differentiation) Made by Miguel Astronomo, Daniel Budo Page 6 of 9  Differentiate Staphylococcus aureus from Coagulase Negative Staphylococcus CoNS SLIDE COAGULASE TEST BACITRACIN SUSCEPTIBILITY TEST (Test for Differentiation) TUBE COAGULASE TEST Differentiate Micrococcus & Rothia from Staphylococcus 0.04 u Bacitracin disk Interpretation: ○ Zones > 10 mm = Susceptible ○ Zones < 10 mm or no zone of inhibition = Resistant Micrococcus and Rothia = susceptible Staphylococcus = Resistant NOVOBIOCON SUSCEPTIBILITY TEST (Test for Differentiation) RAPID DETECTION OF COAGULASE ACTIVITY Latex agglutination Latex particle (coated with human plasma fibrinogen Differentiate S. saprophyticus from other CoNS & IgG) → reacts with clumping factor & protein A of S. saprophyticus = Resistant SAU ○ Other CoNS = sensitive Ex. BBL Staphyloslide Latex Test Place 5ug of Novobiocin Blue Latex reagent + SAU colonies ➔ cross-linking ➔ Interpretation: agglutination w/ latex particle ○ Susceptible = zone > than 16 mm ○ Resistant = zone < or = to 16 mm MODIFIED OXIDSE TEST (Test For Differentiation) DNAse Test (Test for Differentiation) Differentiate Micrococcus from Staphylococcus Reagent: 6% tetramethyl-p-phenylenediamine For the presumptive identification of S. aureus and dihydrochloride in dimethyl sulfoxide differentiates it from CoNS. Result: Methyl green: Colorless ○ (+) = dark blue - purple color (Cytochrome ○ DNA binds with MG = green color C) ○ When DNA is hydrolyzed = freed MG ○ (-)= no change in color (colorless @ pH 7.5) Made by Miguel Astronomo, Daniel Budo Page 7 of 9  1. Cefoxitin Disk Screen Test 2. Latex agglutination for PBP2a 3. MH agar with NaCl and 6 ug/mL of Oxacillin 4. Chromogenic Agar a. MRSA grow and produce mauve-colored colonies 5. Detection of mecA gene or its product PBP2a a. Nucleic acid hybridization & DNA ○ amplificaiton through PCR Toluidine Blue O: bright pink color Treatment and Prevention DNA binds with TBO = structure changes as DNA is For S. aureus: hydrolyzed = bright pink color Handwashing; local antisepsis ○ Detection of thermostable nuclease Drainage of pus Surgical drainage, removal of tissue Antimicrobial Therapy Methicillin, nafcillin, cloxacillin Vancomycin Tetracyclines For S. epidermidis Difficult to treat. Removal of the prosthetics may be done. 40% of CoNS are resistant to ß-lactamase resistant antibiotics Treatment: ○ Penicillin G Detection of Antibiotic Resistant Staphylococcus aureus ○ semisynthetic penicillinase-resistant penicillins ○ cephalosporins ○ Vancomycin For S. saprophyticus The majority of S. saprophyticus infections can be adequately treated with antibiotics. Untreated ➔ progress to pyelonephritis. The antibiotic of choice: ○ Nitrofurantoin (Macrobid) ○ Trimethoprim-sulfamethoxazole (TMP-SMX) Made by Miguel Astronomo, Daniel Budo Page 8 of 9 Made by Miguel Astronomo, Daniel Budo Page 9 of 9