Clinical Bacteriology Past Paper - Emilio Aguinaldo College-Cavite

Clinical Bacteriology BS in Medical Laboratory Science | Emilio Aguinaldo College-Cavite LESSON 1: STREPTOCOCCUS (Dean Basit and Sir Angelo) Taxonomy Numerous taxonomy changes to Streptococcaceae du...

Clinical Bacteriology BS in Medical Laboratory Science | Emilio Aguinaldo College-Cavite LESSON 1: STREPTOCOCCUS (Dean Basit and Sir Angelo) Taxonomy Numerous taxonomy changes to Streptococcaceae due to DNA homology and DNA sequencing studies o Group D (now Enterococcus) Lancefield Classification o Group N (now Lactococcus) Currently, there are 17 genera, 29 recognized species o Streptococcus, Aerococcus, Lactococcus, Leuconostoc, and Pediococcus spp. (resemble streptococci) Lancefield Classification of Streptococci After recognition of the antigen (C Carbohydrate, S. pyogenes (Group A) Antigen Structure polysaccharide – main antigenic characteristics of strep) in the cell wall of B-hemolytic streptococci, these streptococci were able to be LC Group A Streptococci (GAS); cocci in long chains divided into serologic groups designates by letters. M protein attached to peptidoglycan of the cell wall Organisms in the A group possess the same antigenic C Facultative Anaerobe, grows best in 10% CO2 carbohydrate, those in the B group possess the same antigenic Grows on enriched media only carbohydrate, and so on. Resistant to drying, can be recovered from swabs after several hours of collection Streptococci and Enterococci ”Fever-producing” and “Flesh-eating” bacterium, affecting Growth, Gram Stain, and Catalase deeper tissues and organs Most behave like facultative anaerobes Culture (BAP): Grayish-white, small, translucent, smooth Aerotolerant anaerobes – grow in the presence of oxygen but do with a well-defined B-hemolysis not use it for respiration Habitat – Beta Hemolytic Streptococci Pepto streptococcus: Obligate anaerobe This organism is not usually part of a normal human flora as it is Non-motile, non-spore former, non-encapsulated the strain of streptococcus most often associated with disease BAP: Pinpoint – Grow best on enriched media w/ blood, glucose URT: Nose, Throat, Pharynx (causes strep throat) or serum (fastidious – require additional nutrient with blood) Transmission: Direct contact, Fomites Gram-positive cocci seen in pairs or chains, lancet-shaped Biochemical Tests: Oxidase, and Gas production; Non-motile; Virulence Factors Catalase-negative M protein o Can be false-positive if taken from growth media evasion of phagocytosis; attached to peptidoglycan containing blood Anti-complement; Type-specific; hair-like projections Growth and Colony Characteristics For adherence to mucosal cells Some require increased concentration of CO2 (capnophiles) 80 different types (M5 or M10) Stimulates growth of other species is increased in the presence Resistance to infection is related to M protein antibody of CO2 but it is not required production CO2 packet: if you want to achieve 5-10% CO2, expensive Fibronectin-binding protein (Protein F) and Lipoteichoic acid Colonies of growth are usually small and transparent (pinpoint) Adhesion molecules that mediate attachment to host epithelial Grayish, translucent to slightly opaque; Some are mucoid cells and oral mucosa Cell Wall Structures Capsule Thick peptidoglycan layer with teichoic acid (Strep is Gram+) prevents opsonized phagocytosis; non-immunogenic C = carbohydrate layer present except in Viridans group (no LC) a. Allows bacterium to mask its antigens and remain Capsule in S. pneumoniae and in young cultures of most spp. unrecognized by its host’s immune system Hemolysis b. Swollen capsule (Neufeld-Quellung reaction +) Has B-hemolysis, A-hemolysis Streptolysin O Streptolysin S a'-Hemolysis or Wide Zone has small area intact zone of RBCs Oxygen Responsible for hemolysis on blood agar plate with hemolysis around them stable (BAP); antigenic; best to stab the agar to create Y-hemolysis (Gamma-Nonhemolytic) anaerobiosis (Manner of inoculation: Stab and Can lyse RBCs are not lysed, no change in agar color Streak the agar) RBCs and Oxygen labile: only active anaerobically WBCs Staphylococci VS. Streptococci Destroys WBCs, platelets, RBCs, and other Nonantigenic tissues; induces antibody response Responsible Characteristics Staphylococcus Streptococcus for surface Can use anti streptolysin O (ASO) tests to check for recent exposure (Normal: hemolysis on Color in BAP Golden yellow except Grayish BAP S. epidermidis hypersensitivity reaction Post-Strep Sequelae – has two serious complications of GAS Heat-labile, produced by lysogenic strains Rheumatic Fever (Class I M Protein) Clinical Infections o Inflammation of joints, heart, blood vessels, and Strep Throat (S. pyogenes Bacterial Pharyngitis/Tonsilitis) subcutaneous tissues Acute Glomerulonephritis (Class I and II M Protein) Often seen in children 5 to 15 years of age o Occur after pharyngitis or cutaneous infection Incubation period: 1-4 days then abrupt illness ensues o Immunologic mechanisms lead to antigen-antibody Sore throat, malaise, fever, headache complexes, resulting in damage to the kidneys Disease varies in symptoms and intensity; treat with antibiotic to o Immune complex will be deposited at the basement prevent post-streptococcal infections (sequelae, more membrane of our kidneys -> own WBC will attach dangerous than Bacterial pharyngitis) immune complex -> damaged kidneys (constant Symptoms usually subside in 3-5 day unless complications dialysis or death) Transmission: Droplets and Close contact When highly virulent strains appear in schools —> sharp outbreaks of sore throats and scarlet fever Diagnosis: Throat swab Skin infections – may progress to NF, if not treated Skin or Pyodermal Infections – Bites and abrasions to the skin o Impetigo – localized skin disease (young children) ▪ Contagious, close contact w/ infected o Erysipelas – acute infection and inflammation of the Specimen Collection and Recovery dermal layer (painful reddish patches) Specimen Collection ▪ Spreading red rash with demarcated but Swab rubbed over the posterior pharynx and each tonsillar area irregular edge (older adults) (rub it left and right) o Cellulitis – deep invasion of GAS leading to necrosis o Exudate = present, touched w/ swab; exudate is a and gangrene (flesh-eating bacteria) liquid or white patches, result of inflammatory process ▪ Diffuse, spreading infection of Avoid contamination by tongue and uvula subcutaneous skin tissue characterized by erythema (redness) and edema Sepsis – organisms are already in blood (multiplying inside) Gubat, Trisha B. and Saulog, Andrea Bea C. (2023) | MMLS 3-6  Clinical Bacteriology BS in Medical Laboratory Science | Emilio Aguinaldo College-Cavite LESSON 1: STREPTOCOCCUS (Dean Basit and Sir Angelo) Recovery Culture: Grayish white, mucoid colonies, small zone of β- Sheep blood agar (SBA) plate is inoculated and streaked Hemolysis after 24-48 hours of incubation at 35 ̊C (multiple interrupted streak) for isolation o Granada Agar: Yellow to Orange colonies SBA containing Sulfamethoxazole (SMZ) Habitat – Beta Hemolytic Streptococci o Improves recovery of B-hemolytic streptococci from Lower GIT & Female genital tract – normal flora, not normal on throat cultures; SBA w/ SMZ is an inhibitory substance neonates (may result to neonatal meningitis, sepsis, post- Observe after 24 hours for the presence of B-hemolytic colonies puerperal fever or death) o If none, incubate for an additional 24 hours Virulence Factors o a-hemolysis is not clinically significant Traditional and Rapid Detection Methods Capsule – prevents phagocytosis but is ineffective after opsonization Throat cultures: Bacitracin (0.04 unit of Taxo A), Pyrrolidinyl Sialic acid – most significant component of capsule and critical Aminopeptidase (PYR- cherry red), Lancefield Typing (antisera) virulence determinant Other specimens: Hippurate hydrolysis, Christie Atkins Munch- o With mutant strains, Loss sialic acid = Loss virulence Petersen (CAMP) test, Bile Esculin Test, 6.5% NaCl broth – Avirulence Factors: Products Produced by GBS determines the phenotypic characteristics Rapid Detection Tests (RADTs) for GAS Pharyngitis: Hemolysin, CAMP factor (+: with arrow-head hemolysis), o Enzyme immunoassays (EIAs) DNAses, Hyaluronidase, Protease (enzyme that can destroy o Optical immunoassays (OIAs) protein immunoglobulin), Neuraminidase o Rapid-PCR: for genotypic characteristics Clinical Infections Significant cause of invasive disease in newborns Today known as the leading cause of death in infants in the US even though the incidence decreased dramatically from the 1990s to 2008 because of GBS screening Nosocomial transmission: Unwashed hands of the mother or healthcare worker Early Onset Infection Less than 7 days old Group A: Resistant to SXT, Susceptible to Bacitracin 80% of cases vertical transmission (from mother during birth) Group B: Resistant to SXT and Bacitracin Premature birth, membrane rupture, develops into pneumonia or Group A & B: Susceptible to Penicillin meningitis with bacteremia Serological Testing o Results in a very high mortality if not treated quickly o Positive Streptococcal Antigen Test Late Onset Infection o Elevated Streptococcal Antibody Titer (ASO) o Elevated Acute Phase Reactants (ESR, CRP) 7 days old and up (Usually 1 week to 3 months) Commercial Identification Systems Primarily meningitis From blood cultures: β-Hemolytic, Pinpoint colonies Adult Infections Verigene Gram-positive blood culture (BC-GP) nucleic acid test Mother and after childbirth or abortion FilmArray blood culture identification (BCID) o Endometritis or wound infections Matrix-Assisted Laser Desorption Ionization-Time of Flight o Endocarditis (inner wall of heart muscle) (MALDI-TOF) bacterial exact identification systems Older Adults o Immunodeficiency Not all hospitals in the PH have o Skin ad soft tissue infections, intraabdominal abscess, MALDI-TOF, only San Lazaro bacteremia, pneumonia and St. Luke’s have MALDI-TOF. Treatment DEAN Version o Ampicillin with or without an aminoglycoside ANGELO Version o Penicillin and Ampicillin (Most strains sensitive drug of choice) o Penicillin + Ampicillin with Aminoglycosides (Life- threatening cases, Pregnant carriers) o Intrapartum prophylaxis and selective administration antibiotic to infants (90% reduction of sepsis) o Ceftriaxone or Cefotaxime Treatment: Penicillin (Drug of choice), Erythromycin o Vancomycin Prophylaxis: Can prevent RF but not GN Specimen Collection and Handling S. agalactiae (Group B) Antigen Structure Pregnant Women Possess the Group B specific antigen Collect vaginal and rectal swab material between 35- and 37- o Acid-stable polysaccharide in cell wall weeks’ gestation to check if GBS is present o AKA Group B Streptococci (GBS) Inoculate to: and incubate for 18-24 hours Nine recognized capsular polysaccharide serotypes o Todd Hewitt Broth with antimicrobials (Lim Broth) o Three major serotypes: IA, IB, II ▪ Lim Broth (10ug/mL of Colistin +15ug/mL o Contain a terminal residue of sialic acid Nalidixic acid) o Weakly immunogenic and can inhibit activation of o TransVag Broth with 8 ug/mL Gentamicin + 15ug/mL alternative complement pathway Nalidixic acid Gubat, Trisha B. and Saulog, Andrea Bea C. (2023) | MMLS 3-6  Clinical Bacteriology BS in Medical Laboratory Science | Emilio Aguinaldo College-Cavite LESSON 1: STREPTOCOCCUS (Dean Basit and Sir Angelo) o Strep B Carrot Broth: enrichment chromogenic Large Colony Isolates medium in which the presence of Group B S. dysgalactiae, subsp. equisimilis with Group A, C, G, L antigen streptococcus gives the clear medium an orange or o Sometimes type Group A or L red color after 6-24 hours of incubation B-hemolytic (colorless structure) Subculture to SBA (Sheep’s Blood Agar): enriched medium A, C, and G antigens are classified w/ the pyogenic streptococci Morphology and Identification Systems Similar infection sites as Group A strep Grayish-white mucoid colonies surrounded by a small zone of B- Small Colony Isolates hemolysis after 24 to 48 hours incubation at 35⁰C S. anginosus group (included in Viridans) with C and G antigens Gram-positive cocci in chains o Short chains in clinical specimen S. pneumoniae Antigenic Structure o Longer chains in culture Pneumococcus or Diplococcus pneumoniae that have no Presumptive ID based on antigen detection/biochemical reaction Lancefield classification; Contains C substance, similar to C Can use nucleic acid tests (CAMP test) carbohydrate of other Lancefield groups Laboratory Diagnosis Microscopy: Gram Positive, in pairs, short chains, lancet- shaped (pointed ends) or oval, non-motile Facultative Anaerobe; Capnophilic GBS (+) LAP Test Alpha Hemolytic; Fastidious Encapsulated strains (>90%; Virulent); Non- Encapsulated strains (Avirulent) Virulence Factors Capsule – capsular complex polysaccharide; notorious antigen Principal virulence factor Contains 90 known types Basis of Quellung Reaction o Capsule is antigenic and can be identified with appropriate antisera o Quellung Reaction – the capsule swells in the presence of specific anti-capsular serum; also serves to serotype the isolate specifically (serotypes 1-3). Opsonization of the capsule renders the organism avirulent Should be in the nasopharynx and deficient of the specific antibody against the capsule: To contract pneumococcal infection o Opsonization of the capsule renders the organism avirulent. Toxins (Avirulent Factors) Hemolysins, Immunoglobulin A protease, Neuraminidase, CDC Recommendations Hyaluronidase Universal vaginal and rectal screening of all pregnant females Clinical Infections late in the 3rd trimester GBS Screening pregnant women with asymptomatic bacteriuria Normal flora in Upper respiratory tract (URT) and as a pathogen Prophylactic antimicrobial therapy for women whose samples Pneumococcal (Lobar) Pneumonia with test positive results: Prevalent in older adults and immunocompromised individuals o Vaginal and rectal screenings Occurs as secondary infection (Alcoholism, anesthesia, o Urine culture malnutrition, or after viral infection) o Women with previous history Has been identified as an etiology of the Community Acquired o An unknown status of GBS at time of delivery Pneumonia (CAP) in adults and a major cause of Healthcare Group C, F, and G Streptococci Associated Pneumonia (HCAP) Recovered from URT, Vagina, and Skin of humans Bacterial meningitis in adults Possess M Protein (like Group A streptococci) Colonizer of Nasopharynx of healthy individuals (5-75%) Isolates of this group with beta hemolysis are detected by o It is an asymptomatic member of a normal respiratory serotyping with latex agglutination reagents tract such as the nasopharynx Associated with Pharyngitis and Skin infections (Impetigo) Infection begins with aspiration of organisms Transmission: Person-to-Person contact Chills, cough, dyspnea (shortness of breath) Group C streptococci: Main source of streptokinase Leads to edema of the lungs and drowning in own fluids Mostly animal pathogens High mortality even w/ treatment (5-10%), 50% w/o treatment In humans: S. dysgalactiae subsp. equisimilis Pass thru the In horses: S. equi subsp. zooepidemicus BBB -> CNS Beta-hemolytic except S. dysgalactiae Express protein G (surface of bacteria) Middle ear Culture: Small, flat, grayish-white with regular to narrow zone of infection “LUGA” alpha or beta hemolysis Antimicrobial Susceptibility Testing: o Group C streptococci – Susceptible to Bacitracin and SXT Can also be involved in o Group G streptococci – Bacitracin variable o Endocarditis (inflammation of heart muscle) o Peritonitis (all from dissemination) Gubat, Trisha B. and Saulog, Andrea Bea C. (2023) | MMLS 3-6  Clinical Bacteriology BS in Medical Laboratory Science | Emilio Aguinaldo College-Cavite LESSON 1: STREPTOCOCCUS (Dean Basit and Sir Angelo) o Hemolytic Uremic Syndrome (caused by E. coli Other available tests O157:H7) Phadebact Pneumococcus Test (for CSF; can test for Neisseria ▪ Blood and CSF positive meningitidis, Thermophilus influenzae, Strept. B, pneumococcus) ▪ False-positive Coombs test (direct MALDI-TOF MS antiglobulin test in blood banking- Now S. pneumoniae antigen card – for antigen detection in urine complicate cross-matching result) FilmArray BCID Panel – for ID in blood cultures Transmission: Direct contact, Droplets, Secretions by infected CDC Recommendations on Vaccination: Antimicrobial Resistance and Vaccination o 13-valent Conjugated vaccine (PCV13): 2-month- Penicillin (If Resistant, use Erythromycin or Chloramphenicol) old as part of the pediatric immunization Vaccine (Prevenar, FluVax) – to prevent secondary infection o 23-valent Polysaccharide vaccine (PSV23): adults o Against most common capsule antigens o >65 years old: Given upon the advice of clinician o Recommended for asplenic individuals, older adults, cardiac patients o Helps reduce incidence and severity Laboratory Diagnosis Gram-positive diplococci with lancet shape (lanceolate) As culture ages, Gram-stain reaction becomes variable (false -) o Sputum/Secretions, CSF Capsule stain (can reveal the capsule – halo-like) Culture Media and Colony Appearance Test to Differentiate Pneumococci Culture Pneumococci Other Streptococcus Complex media required such as brain-heart infusion (BHI), SBA, Bile Solubility Bile soluble Insoluble or chocolate agar with increased CO2 Inulin Fermentation Fermenter Non-fermenter Typical large zone of a-hemolysis on blood agar surrounding the colonies; with Taxo P (S. pneumoniae is susceptible to Optochin Neufeld Quellung Capsular swelling No swelling Test); active component of Taxo P is ethylene hydrocupreine Quinidine Susceptible Resistant Young Cultures Optochin Susceptible Resistant Round, glistening, wet, mucoid, dome-shaped appearance Mouse Virulence Mouse dies within 16- Mouse alive Test 48 hours Older Colonies Autolytic changes result in a collapse of each colony’s center giving it the appearance of a coin with a raised rim; dimple- shaped, donut-shaped colonies Identification Methods Traditional tests Bile solubility (S. pneumoniae is lysed by bile-positive; S. mitis not lysed by bile-negative) Optochin susceptibility Viridans Streptococci Normal Flora: URT, Female genital tract, and GIT, Oral cavity, Nasopharynx (oropharyngeal commensals Lack of Lancefield group antigens (lack of M protein) Many shows a-hemolysis (alpha prime) or nonhemolytic Fastidious, with some requiring CO2 for growth Viridans streptococci 5 groups (Some may have A, C, G, or N Lancefield antigen) o S. mitis, S. mutans (anaerobic-dental caries), S. salivarius, S. bovis, S. anginosus Virulence Factors Specific species within this group, for example, S. mutans, S. sanguis are able to form biofilms on tooth surfaces (plaque) Fermented carbohydrates = Acid metabolites are then important in the formation of dental caries (tooth decay) Gubat, Trisha B. and Saulog, Andrea Bea C. (2023) | MMLS 3-6  Clinical Bacteriology BS in Medical Laboratory Science | Emilio Aguinaldo College-Cavite LESSON 1: STREPTOCOCCUS (Dean Basit and Sir Angelo) First degree caries affects the enamel Newer Identification Methods Not well established Development of molecular methods for identification over A polysaccharide capsule and cytolysin have been identified in Viridans streptococci has been challenging (DNA hybridization, some members of the Anginosus group PCR); MALDI-TOF systems are in the evaluation stage Other factors with possible virulence tendencies Multilocus sequence analysis may be used to identify unknown o Extracellular dextran, cell surface-associated proteins, streptococci species extracellular enzymes Treatment Clinical Infections Penicillin with or without Aminoglycosides For immunocompromised people, Viridans are significant Ceftriaxone Most common cause of subacute bacterial endocarditis (SBEs) Vancomycin (Resistant strains and Allergic to Penicillin) o More common in children than in adults with Laboratory Diagnosis hematologic malignancies ▪ Heart valves damaged by rheumatic fever B-D-Glucuronidase Test (BGUR) are especially prone (people with GAS) Differentiates the B-Hemolytic streptococci in larger colony Other diseases: Tooth decay, meningitis, abscesses, group C and G from the small colony group C and G of the S. osteomyelitis, and empyema (gain entry on dental procedures) anginosus group or milleri complex Most penicillin susceptible Diagnostic Tests for S. bovis group (Group D streptococci) These opportunistic organisms are of low pathogenic potential Growth in Bile Esculin Medium: One of the few instances in which they cause disease is when o Reagents: Esculin and 1-4% Bile salt they are able to infect previously damaged heart valves and o (+): Black color complex in the agar within 48 hrs. cause endocarditis 6.5% NaCl (Nutrient broth base) test: No growth (Negative reaction/Absence of turbidity) PYR Test: Negative (No color change) Penicillin Test: S.bovis susceptible to Penicillin Viridans Streptococci Group D Versus Enterococci S. bovis group and enterococci both possess the D antigen o S. bovis is no longer a valid species name S. bovis and S. equinus are the same species Both groups are bile-esculin positive o Nonhemolytic enterococci were 6.5% NaCl negative (Salt tolerance test) o Enterococci were 6.5% NaCl positive Enterococcal group is now in genus Enterococcus whereas Non enterococcal group is part of D streptococci (GDS), characterized by UTI, cardiovascular infection, and meningitis Group D sometimes manifests infections (endocarditis, UTI, wound infection or abscess) Distinguish Group D from Enterococcus o Enterococcus = Resistant to Penicillin o Group D = Susceptible to Penicillin Differentiation of Nonhemolytic and a-Hemolytic Streptococci Laboratory Diagnosis from Enterococci Extremely difficult to identify isolates Laboratories should be satisfied to place isolates into one of the five groups Typical characteristics on Gram Stain Culture colonies are small and surrounded by a zone of a- hemolysis (Some strains may either B-hemolytic or nonhemolytic) All members are PYR negative and Leucine aminopeptidase (LAP) positive Colonies: Minute to small gray, domed, smooth or matte, alpha (some non) hemolytic Enterococci (GDS) Optochin Test: Resistant Bile Esculin Medium – Positive: Black color complex in agar Microbiota: Human and animal intestinal tracts, female GUT Penicillin test: Susceptible Two most Common Species: E. faecalis and E. faecium Bile: Insoluble All species produce D antigen and thus belong to the Lancefield Growth 6.5% NaCl: No growth at high alkalinity D group (GDS) Identification Systems Pseudo catalase Reaction: Weak bubbling in catalase test Can grow under extreme conditions like bile, salt, or alkaline pH Virulence Factors Resistant to multiple antimicrobial agents Surface adhesion proteins Extracellular serine protease Gelatinase Two subunit toxins called Cytolysin in E. faecalis Gubat, Trisha B. and Saulog, Andrea Bea C. (2023) | MMLS 3-6  Clinical Bacteriology BS in Medical Laboratory Science | Emilio Aguinaldo College-Cavite LESSON 1: STREPTOCOCCUS (Dean Basit and Sir Angelo) Clinical Infections Clinically Significant Streptococcus-like Organisms (SLO) Nosocomial Infections – opportunistic pathogens UTI is the most common, often associated with urinary catheterization or other urologic manipulations Prolonged hospitalization Bacteremia, wound infection Endocarditis (enterococcus 5% to 10% of bacterial endocarditis) Growth Criteria and Characteristics Standard collection and handling procedures Specimens should be cultured ASAP Resistant to multiple antimicrobial agents with temperature- dependent growth properties Transmission: Direct contact, Fomites, Contaminated medical instruments Gastrointestinal carcinoma: S. gallolyticus subsp. gallolyticus in blood cultures Laboratory Diagnosis Abiotrophia and Granulicatella AKA Pyridoxal-Dependent; Vitamin B6-Dependent; Thiol- Dependent; Symbiotic Strep Differential Tests: Disease: Bacteremia, Endocarditis, Otitis Media, Brest-implant- - (+) Tellurite Test: associated infections, Endophthalmitis, and Septic Arthritis E. faecalis Part of the human oral and gastrointestinal microbiota - (+) Arabinose Thiol compounds: Cysteine, vitamin B6 and pyridoxal Formation: E. faecium Difficult to treat with antibiotics Formerly Nutritionally-Variant streptococci (NVS); gram variable o Requires sulfhydryl compounds or 10mg/L of Pyridoxal Hydrochloride added to media (BAP or CAP) Trypticase soy or BHI agar supplemented with 5% Sheep Blood Culture: Grow as “satellites” around bacteria that produces o Recommended Media for Contaminated Specimens: Pyridoxal such as S. aureus (“Staphylococcal streak test”) Bile Esculin Azide Agar, CNA, PEA, Cephalexin o Resemble Viridans streptococci Aztreonam - Arabinose agar o Abiotrophia will not grow on BAP or CAP unless o Colonies are creamy, white, glistening, smooth with Pyridoxal is supplied varied hemolysis o Exhibit alpha or gamma hemolysis o E. faecalis: Identified by Its ability to grow in the Laboratory test: staphylococcal streak test; (+) PYR and LAP Presence of Tellurite Microscopy: Gram variable, pleomorphic Bile Esculin Test: Bile Resistant —> Black color Aerococcus - airborne 6.5% NaCl —> Turbidity (Pink —> Yellow) Opportunistic pathogen Penicillin Test: Resistant o Bacteremia, endocarditis, UTI in immunocompromised people Resembles Viridans streptococci on culture Resembles staphylococci on Gram Stain o Gram-positive cocci in tetrads or clusters o Weak catalase (aka pseudo catalase reaction) o Susceptibility patterns similar to enterococci Common isolates: Antimicrobial Resistance of Enterococcus o A.viridans: (+) Bile Esculin and PYR Test Acquired resistance to aminoglycosides, B-lactams, and o A.urinae:(-) Bile Esculin and PYR Test glycopeptides Urinary Tract Pathogens: A. urinae and A. sanguinicola Resistant to cephalosporins and some aminoglycosides like Gemella Gentamicin and Erythromycin Similar morphology with Neisseria spp.: Diplococci with adjacent Enterococci are resistant to Efromycin Acid Disk (100ug) test sides flattened Vancomycin-Resistant Enterococci (VRE) Either produce a-hemolysis or are nonhemolytic Predominantly E.faecium due to the resistant genes known as o Species: G. haemolysans, G. morbillorum, G. bergeriae, G. sanguinis the van genes(VanA, VanB, VanC) o Common Isolates: G. haemolysans (Aerobic) and G. Eight phenotypes have been desired morbillorum (Anaerobic) VanA and VanB most frequently encountered Decolorize easily during Gram staining (mistaken as Gram-) o VanA is highly resistant to vancomycin (located in o Often appear as gram-negative cocci in tetrads, pairs, plasmids and transposons) clusters, or short chains Vancomycin-containing agar and chromogenic VRE media have Biochemical Tests: (+) PYR, (-) Bile Esculin, Growth in 6.5% been used for screening NaCl broth PCR-based assays have been used for ID Common isolates are susceptible to Penicillin Gubat, Trisha B. and Saulog, Andrea Bea C. (2023) | MMLS 3-6  Clinical Bacteriology BS in Medical Laboratory Science | Emilio Aguinaldo College-Cavite LESSON 1: STREPTOCOCCUS (Dean Basit and Sir Angelo) Lactococcus (formerly GNS) Place streptococci into a dilute acid solution and heat for 10 mins. Gram-positive cocci singly, in pairs, or in chains Soluble antigen is used to immunize rabbits Physiologically similar to enterococci but do not produce acids Rabbits develop antibodies to the antigen from carbohydrates Classified several different antigens (carbohydrate groups) Has been isolated from dairy and plant products A, B, C, D, F, G (Group F and G are animal pathogens) Are either a-hemolytic or nonhemolytic, may grow at 10 ̊C Hemolytic Lancefield Amino group – Species Associated with UTI and endocarditis Reaction Group Specific CHO Leuconostoc (Gram +) α None ------------ S. pneumoniae Irregular coccoid morphology β- A Rhamnose-N- * S. pyogenes Share phenotypic and biochemical characteristics with hemolytic acetylglucosamine Lactobacillus spp., Viridans streptococci, Pediococcus spp., and B Rhamnose- * S. agalactiae Enterococcus spp. ------------ Glucosamine Gas producer by MRS (Mann, Rogosa, and Sharp) method with polysaccharide alpha or beta hemolytic pattern C, G Rhamnose-N- Acetyl S. dysagalactiae Species: L. citreum, L. dextranicum, L. lactis, L. mesenteroides ------------ galactosamine subsp. Associated with bacteremia, UTIs, meningitis, pulmonary inf. equisimilus Intrinsically resistant to vancomycin Glycerol Teichoic γ D S. bovis Laboratory Tests: (+) Bile Esculin hydrolysis and growth in 6.5% Acid containing D- NaCl broth; (-) PYR and LAP Alanine and Glucose α, β or γ D Glycerol Teichoic Enterococcus Pediococcus Acid containing Alanine Isolated from natural environment like vegetables and Glucose Arranged in tetrads, pairs, and clusters α and γ None ------------ Viridans Resembles streptococci or enterococci Bacteremia, abscess, meningitis Small colony Anginosus group Intrinsically resistant to vancomycin similar to Leuconostoc ------------ variants of A, ------------ C, F, G or Associated with infections ungroupable o Patients who have underlying GI abnormalities or previously had abdominal surgery This sometimes show isolates that are nonhemolytic. o Linked to bacteremia, abscess formation, meningitis Summary of Classification Species: P. acidilactici and P. damnosus Bacteria Smith and Lancefield Academic Laboratory Test: (+) BE and LAP; (-) PYR Brown Laboratory Diagnosis S. pyogenes Beta Group A Pyogenic Smith + Brown Classification S. agalactiae Beta Group B ------------ – hemolytic pattern on sheep blood agar (SBA) S. dysgalactiae Beta Group C Pyogenic subsp. equimius S. bovis Alpha, Gamma Group D ------------ Enterococcus Mostly Gamma Former: Group Enterococcus D Viridans Alpha, Gamma None Viridans Physiologic Characteristics (Bergey’s Classification) S. pneumoniae Alpha None ------------ – based on temperatures; all can grow at body temperature (37⁰C) Pyogenic Streptococci – produces pus, mostly B-hemolytic Laboratory Diagnosis for Lancefield Groups of Streptococci and constitute most of the Lancefield groups, cannot grow at 45⁰C and 10⁰C o S.pyogenes, S.agalactiae, S.dysgalactiae subsp equisimilis, S.anginosis group Lactococci (Lactic) – nonhemolytic with Lancefield Group N antigen; often found in dairy products (S. lacticola/S. lactis – causes normal coagulation or souring of milk); cannot grow at 45⁰C but can grow at 10⁰C Enterococci – part of normal intestinal biota; GIT; can grow at 45⁰C and 10⁰C Viridans streptococci – widely found in URT; can grow at 45⁰C but not at 10⁰C Surface typing (Lancefield classification)– serologic typing of C carbohydrate, capsular polysaccharide, surface protein Biochemical characteristics Key tests to determine presumptive identification o Hemolytic reaction of the isolate dictates which select identification tests should be done Exhaustive testing not always needed Lancefield Classification Scheme Rebecca Lancefield developed this technique in 1930s Gubat, Trisha B. and Saulog, Andrea Bea C. (2023) | MMLS 3-6  Clinical Bacteriology BS in Medical Laboratory Science | Emilio Aguinaldo College-Cavite LESSON 1: STREPTOCOCCUS (Dean Basit and Sir Angelo) RapID STR Panel for Identification of Streptococcus spp. In modified VP test, a heavy suspension of bacteria is incubated in 2 mL VP broth (6 hours) Add a few drops of 5% α-naphthol and 40% potassium hydroxide o Shake to increase dissolved oxygen o Incubate 30 minutes at room temperature Red or pink is positive (e.g., Anginosus group) B-D-Glucuronidase (BGUR) Test Detects action of B-glucuronidase o Enzyme found in isolates of large-colony-forming Group C and Group G B-hemolytic streptococci o Not found in small colony isolates Commercially prepared rapid assays are available Bile Esculin Hydrolysis Test Enterococci is +; ability to grow in 40% bile and hydrolyze esculin are features of streptococci that possess Group D antigen Salt-Tolerance Test Growth in 6.5% NaCl broth o Differentiates Group D streptococci from enterococci o Enterococci grows; Group D do not Bacitracin Susceptibility Optochin Susceptibility Test Susceptibility Tests – Bacitracin (0.04 units) or “A” disk Optochin (Taxo P Disk) – Ethylhydrocuprein hydrochloride o Identifies GAS – susceptible to A disk Disk is added to surface of SBA just inoculated with an a- PYR disk as a better test because reaction is more definitive hemolytic Streptococcus CAMP Test Overnight incubation then examines for zone of inhibition Three-ways to perform the test o Zone sizes (Positive and Susceptible) 1) Use of B-lysin producing S. aureus ▪ Greater than 14mm with 6-mm disk (+; S) 2) Use of a disk impregnated with B-lysin ▪ Greater than 16mm with 10-mm disk (+; S) 3) Rapid CAMP test (aka spot CAMP test); screen for GBS Presumptive identification of S. pneumoniae Principle: Detects the production of enhanced hemolysis Bile Solubility Test Occurs when B-lysin (S. aureus) and the hemolysins of Group B Takes advantage of the diagnostic for S. pneumoniae streptococci come in contact autocatalytic enzyme amidase o Also, can use purified B-lysin on confluent strep colony o Under influence of bile salt or detergent cell wall lyses o Disks containing B-lysis also work during cell division o Group B streptococci showing the classical “Arrow- o Suspension of colony in sodium deoxycholate shaped (+)” hemolysis near the staphylococcus streak ▪ Clearing through lysis of colonies Hippurate Hydrolysis Test Noncultural Identification Differentiates Group B streptococci from other B-hemolytic Immunoassays streptococci Group B streptococci possess the enzyme Hippuricase Direct clinical specimens (Hippurate hydrolase) which hydrolyzes sodium Hippurate o Throat swab forming sodium benzoate and glycine Isolated colonies Add Ninhydrin reagent to get purple complex (+ for HH Test) o Extract C carbohydrate ▪ Use in agglutination test L-Pyrrolidonyl-a-Naphthylamide Hydrolysis (PYR) Examples of other available immunoassays Presumptive ID of GAS and nonhemolytic Group D streptococci o Latex agglutination Streptococcus pyogenes (GAS) is the only streptococcus o Slide agglutination species that is PYR-positive o Enzyme-linked immunosorbent assay (ELISA), OIA Other PYR-positive: Enterococcus, Gemella, Aerococcus Nucleic Acid Probes – more expensive test Real-time PCR o Detect genes for specific groups ▪ Rapid results and increased specificity DNA probe tests o AccuProbe (16S rRNA for prokaryotes) ▪ Detects pneumococcus Leucine Aminopeptidase (LAP) Test Susceptibility Testing Smear organism on damp LAP disk Penicillin is the drug of choice o Incubate for 5 minutes o Resistant isolates have been reported o Hydrolysis of Leucine B-naphthylamide o Incidence of penicillin-resistant strains on the increase o Releases B- naphthylamide Possible alternative choices: Erythromycin and Narrow- Positive Result: Red after adding p- Spectrum Cephalosporins dimethylaminocinnamaldehyde (DMACA) reagent Multidrug-resistant strains have been reported (S. pneumoniae) Useful in differentiating along with PYR test Vancomycin resistance increasing (VRE) o Aerococcus and Leuconostoc (Negative) Select combinations of drugs can be effective and are based on Voges-Proskauer (VP) Test susceptibility patterns and body sites of specimen origin Detects acetoin production from glucose; biochemical test Gubat, Trisha B. and Saulog, Andrea Bea C. (2023) | MMLS 3-6  Clinical Bacteriology BS in Medical Laboratory Science | Emilio Aguinaldo College-Cavite LESSON 4: STAPHYLOCOCCUS (Dean Basit and Sir Angelo) Micrococcus (Gram +) Coagulase positive staphylococci Micrococcus luteus S. aureus – major human pathogen Gram-positive, catalase positive; non-pathogenic S. delphini, S. intermedius, S. hyicus, S. shleiferi – animals o Modified catalase containing a mild detergent Coagulase negative staphylococci (CONS) – has slime factors Coagulase negative (enzyme that coagulate plasma) S. epidermidis – hospital acquired infections/nosocomial Distinct yellow pigmented colony; nonpathogen S. saphrophyticus – UTIs in young sexually active females Staphylococcus (Gram +) o Important in urine samples General Characteristics Causes infection when it enters a normally sterile site (trauma or abrasion of the skin or mucosal surface) Spherical, non-motile, grape like clusters (Strept are in chains) No capsule, non-spore former; Staphylococcaceae (1 um) Extracellular Factors (Cytolytic Toxins) Aerobic and facultative anaerobe 1. Cytolysin or Hemolysin o S. saccharolyticus (obligate anaerobe) Exotoxin, membrane damaging, tissue destruction and abscess Strongly catalase positive (+) Destroys RBCs and targets eukaryotic cell membranes (Causes o Catalase: can hydrolyze hydrogen peroxide (H2O2) anemia, makes Iron available for microbial growth) into water and oxygen ▪ Staphyle- bunch of grapes 2. Alpha Hemolysin ▪ Kokkos- berry Predominant Hemolysin produced by S. aureus: Act on broad Can ferment glucose except S. saprophyticus spectrum of eukaryotic cell membranes Morphology (Colonial characteristics in a culture plate) Dermonecrotic activity: Severe tissue damage, destroys RBCs and Platelets Appear creamy white, or rarely light “gold” = aureus 3. Beta Hemolysin Buttery looking; Oil paint appearance (S. aureus) Some species produce ᵝ-hemolysis (S. aureus) Degrade sphingomyelin around nerves (Sphingomyelinase C); Colonies (4-8 mm) on blood agar plate appear creamy, white or Destroys RBCs light gold, and with butyrous appearance (“buttery looking”) Hot-cold lysin (works best at 37 ̊C, very well when stored at 4 ̊C) Other species have gray colonies (Staphylococcus epidermidis) 4. Delta Hemolysin Resistance to drying, heating at 50 C for 30 minutes, 10% NaCl Disrupt biological membrane by detergent-like actions Epidemiology Causes injury to cells and Leukocytes but is less lethal Ubiquitous – indigenous microbial flora Has a possible role in S. aureus diarrheal diseases Chief sources of infections: 5. Gamma Hemolysin o Discharge human lesions Leukocidin (Lyses WBCs); Less toxic than Alpha and Beta Lysin o Respiratory tract Interact with Panton-Valentine Leukocidin (capable of causing o Fomites contaminated with discharge pore formation in the cell membrane – increases cation o Skin permeability) o Hospitals – NICU, ICU, OR Produced by all S. aureus strains that cause RBC injury in culture Normal flora of the skin, respiratory tract, and intestines and produces edematous lesions Predisposing factors o Immunosuppression o Concurrent diseases o Antibody resistance Staphylococcus aureus Habitat: anterior nares/nose (20%-30% of human are carriers) Primary pathogen of the genus; 7.5-10% NaCl (halophilic) Produce superficial to systemic infectious (skin, bacterial sepsis) Mannitol Salt Agar: Golden yellow pigment K Tellurite Medium: Jet black colonies Blood Agar Plate: ᵝ-hemolytic S. citreus – lemon yellow pigment S. albus – porcelain white pigment Mode of Transmission Traumatic introduction (needle stick, burns, road rash, medical) Predisposing conditions Chronic infections, skin injuries, indwelling devices Virulence Factors of S. aureus Immune response defects (high risk to immunocompromised) 1. Enterotoxins – staphylococcal food poisoning Coagulase Heat-stable exotoxins that cause diarrhea and vomiting 2. Toxins A-E and G-I (Eight total, no F) Enzyme produced by S. aureus that clots plasma; principal Resistant to gastric acid virulence factor o B and C, sometimes G and I = toxic shock (↓BP) Coagulase Test: single best criterion to determine that 3. Toxic Shock Syndrome Toxin-1 (TSST-1 or Toxin F) pathogenicity of Staphylococcus Produced by phage group 1 causing toxic shock syndrome Promotes formation of a fibrin layer around the staphylococcal o Transduction abscess, protecting the bacteria against phagocytosis o TSST-1 and B, C, G, and I are superantigens (Fibrinogen in Plasma) (microbial peptides: can contribute to autoimmune disorder) Gubat, Trisha B. & Saulog, Andrea Bea C. (2023) | MMLS 3-6  Clinical Bacteriology BS in Medical Laboratory Science | Emilio Aguinaldo College-Cavite LESSON 4: STAPHYLOCOCCUS (Dean Basit and Sir Angelo) 4. Exfoliative toxin (Epidermolytic Toxin) Causes sloughing off of the skin and is known as o Staphylococcus Scalded Skin Syndrome (SSSS) o Ritter’s disease ▪ Associated with bullous impetigo 5. Leukocidins (Panton-Valentine Leukocidin) Kills PMN leukocytes to prevent phagocytosis Pore-forming exotoxin that suppresses phagocytosis Responsible for necrotizing skin and soft tissue infections 6. Hemolysins (Cytotoxic Toxins) – lyse RBC; ᵞ is not important a- Hemolysin: destroys platelets and tissues; incomplete h. o RBCs are partially lysed b- Hemolysin: shows enhanced activity by acting on the sphingomyelin (intact) of RBC membranes causing lysis o Hot-cold lysin since it works best at 37⁰C and very well when stored at 4⁰C o Complete hemolysis, clear area around colony Types of Hemolysis d-Hemolysin: causes injury to cells and leukocytes but is less lethal Blood agar plate = blood agar base + 5-10% sheep’s blood 7. Enzymes o If sheep’s blood is not available, use horse RBC Coagulase: very diagnostic but not completely understood o If no horse RBC, use expired human blood from BB ▪ Not ideal because it may have antibiotic Hyaluronidase (Spreading Factor): hydrolyzes hyaluronic o If you heat the blood => brown (Chocolate Agar plate) acid, present in connective tissues helping spread of o Enriched, support fastidious organisms (nutrients) infection Alpha: incomplete hemolysis (Greenish brown) Lipase: breakdown of the fats and oil created by the sebaceous glands on skin surfaces (pimple-causing) Beta: complete hemolysis (Colorless) 8. Protein A Gamma: no hemolysis (Red cells are still intact) Immunologically active substance: Major protein Epidemiology of S. aureus component of the cell wall Primary reservoir: Nares Group specific antigen– only for S. aureus Other reservoirs: Axillae, vagina, pharynx, and other skin surface Bind to the Fc portion of antibodies to avoid phagocytosis Hospital outbreaks (nosocomial): Nurseries, Burn units, Surgical o Masking of its immunogenic proteins with host o 2nd to E. coli as causative agent proteins to look like “self”; block phagocytosis Infections of S. aureus o Assists in blocking phagocytosis o Competes with neutrophils in the Fc portion of CUTANEOUS INFECTIONS specific opsonins Pus formers – pyogenic, nana -> Fatal septicemia TOXINS Summary Furuncle (Boil/Pigsa) o A painful inflammation of the skin and subcutaneous - Food Poisoning (Gastrointestinal upset —> Food Poisoning), Heat-stable (Heating at 100 ̊C for 30 minutes; tissue; single draining enlarged furuncle reheating contaminated food does not eliminate the o Deep seated infection, originating from folliculitis Enterotoxins organisms) o Large, raised, superficial abscess A, B, C1, C2, - Appears to act as neurotoxins that stimulate vomiting o Commonly found on neck, armpit, and groin regions D, E, G to J through the vagus nerve Carbuncles - Resistant to Hydrolysis by Gastric and Jejunal enzymes o Boils that have multiple lesions and may progress into - Presentation: More vomiting than diarrhea, stimulation of deeper tissues; redness, swelling, fever, chills CNS (Vomiting center) (systemic infection) Enterotoxin - Most common, acts on vascular smooth muscles o May require surgical intervention A o Aggregation of infected furuncles; may form large - Majority of food poisoning (Together with D) abscess Enterotoxin - Damage intestinal epithelium (Pseudomembranous colitis) Folliculitis – hair follicle infection; small red bump on follicles B - Found in contaminated milk products Sty – type of folliculitis affecting one or more hair follicles, edge of upper or lower eyelid Enterotoxin - Responsible for Staphylococcal food poisoning, Inhibits A, B, & D water absorption Staphylococcal Impetigo - Source of contamination: Infected Food handler o Produce watery blisters that will become pustules or sores on the face, neck, hands, and diaper area - Acts on the vomiting center of the nervous system o Large pustules surrounded by small zone of erythema - Enterotoxin A: Most common etiology (redness) Enterotoxins Associated with Enterocolitis o Spread direct contact and fomites – highly contagious B, C, G, I o S. aureus causes bullous and non-bullous impetigo Wound Infections – from burns Abscess – Typical lesion of S. aureus (Hallmark) Necrotizing Fasciitis: inflammation of the fascia (skin and muscle); common on Strep “flesh-eating bacteria) TOXIN-INDUCED CASES Staphylococcus Scalded Skin Syndrome (Ritter’s disease) o More likely to occur in renal failure patients and immunocompromised patients o Exfoliative Disease Gubat, Trisha B. & Saulog, Andrea Bea C. (2023) | MMLS 3-6  Clinical Bacteriology BS in Medical Laboratory Science | Emilio Aguinaldo College-Cavite LESSON 4: STAPHYLOCOCCUS (Dean Basit and Sir Angelo) o Former Name: Dermatitis Exfoliativa, Pemphigus Staphylococcus aureus complex neonatorum, Lyell’s Disease, Ritter’s Disease Members: Staphylococcus aureus, Staphylococcus argenteus, o Mild (localized lesion) Severe (large generalized area Staphylococcus schweitzeri with profuse peeling of epidermal layer Characteristics: o Carries by bloodstream to epidermis split cellular layer o S. argenteus and S. aureus: tube coagulase-positive, o Occurs primary in newborns, previously on healthy PYR negative children o S. argenteus found to be carrying resistance genes o Lasts for 2-4 days such as blaZ (most predominant), erm(C), msr(A), ▪ Recovery on children, Mortality on adults lnu(A), and aac(6’)-Ie-aph(2”)-Ia Toxic Epidermal Necrolysis (TEN) o S. argenteus and S. schweitzer are said to be isolated o Multiple causes: Drug-induced, Infections, Vaccines from bats (cause not known) Differential Biochemical Tests o Similar to SSSS, but TEN responds well to steroids o D-Ribose Fermentation: (+) S. schweitzeri o High mortality rate o N-Acetyl-D-Glucosamine: (+) S. aureus Toxic Shock Syndrome (related to Toxin F) o D-Galactose Fermentation: (+) S. aureus and S. o Associated with super absorbent tampons argenteus o High fever, Rash of trunk spreading to extremities, Coagulase Negative Staphylococci (CONS) Watery diarrhea, Vomiting (will cause dehydration), Staphylococcus epidermidis group hypotension, conjunctival reddening, kidney failure o DIC – disseminated intravascular coagulation o S.epidermidis (presence of coagulase in vivo); significant o S.haemolyticus consequences of toxic shock syndrome; D-dimer test o S.capitis o Increase in BUN and creatinine (renal function test) o S. hominis o Fatal in 2-5% of cases due to multiorgan system o S. pasteuri failure o S. auricularis o Rare but potentially fatal (shock) o S. saccharolyticus Food Poisoning o S. warneri o Caused by toxin not bacterial growth o S. caprae Staphylococcus saprophyticus group ▪ Enterotoxin A to D (A and D most common) ▪ From rich foods (Mayonnaise, potato salad) o S.saprophyticus ▪ Inadequate refrigeration o S.equorum ▪ Enterotoxin are heat-stable o S.nepalensis o Symptoms: appear 2-8 hours after ingesting food o S.cohnii Staphylococcus intermedius group ▪ Resolve in 24-48 hours, sometimes less (Self-limiting) o S.schleiferi subsp.schleiferi ▪ Nausea, vomiting, abdominal pain, cramps Staphylococcus stimulans group o Foods Involved: Salad, Bakery products, Milk and o S.stimulans Dairy products, Meat, Poultry and Egg products Staphylococcus sciuri group o Caused by unhygienic preparation of foods o S.sciuri DEEP INFECTIONS o S.lentus Others: Secondary Pneumonia CONS Unspecified group: S. lugdunensis o After Influenza A (H1N1) but relatively rare ▪ Has high mortality rate Novobiocin Susceptible Novobiocin Resistant Other: Bacteremia S. epidermidis S. saprophyticus o IV-drug addicts with fever S. haemolyticus S. cohnii o Enter through injection site o Treat with Daptomycin w/ or w/o B-lactams (MRSA) S. capitis S. kloosii Other: IV-drug addicts with fever S. lugdunensis S. xylosis o Enter through injection site S. saccharolyticus o Inflammation of inner layer of heart (Endocardium) S. hominis subsp. hominis o Bacteria in bloodstream to heart and lodge on S. warneri abnormal heart valves or damaged heart tissue Osteomyelitis Staphylococcus epidermidis - CONS o Occurs to secondary to bacteremia and results in bacteria invading the bone Former Name: Staphylococcus albus o Fever, chills, swelling, pain, deformity, pus, defect heal Nosocomial infections Deep Organ Abscess – brain, kidney, eyes, lings, spleen, liver, o Hospitalized patients: Impaired host resistance CNS o *Skin flora gets introduced in catheters (indwelling an Periostitis IV), prosthetic heart valves, CSF shunts o Inflammation of periosteum o Produces slime layer (biofilm) that helps adherence to o Fever, localized pain, leukocytosis prosthetics and avoidance of phagocytosis; UTIs Arthritis if bacteria in the joint (septic arthritis) o Secretes an Exopolysaccharide and a Delta Exotoxin Sometimes occur due to block follicles, sebaceous glands, and that contributes to its virulence sweat glands. Cystic Fibrosis, Suppurative Intracranial Phlebitis *Skin, Respiratory Tract and GIT – resident flora Methicillin-resistant Staphylococcus aureus (MRSA): Key health Coagulase Negative, Novobiocin Sensitive alert (Acquired or Nosocomial) Stitch abscess, mild UTI, endocarditis, bacteremia, meningitis Gubat, Trisha B. & Saulog, Andrea Bea C. (2023) | MMLS 3-6  Clinical Bacteriology BS in Medical Laboratory Science | Emilio Aguinaldo College-Cavite LESSON 4: STAPHYLOCOCCUS (Dean Basit and Sir Angelo) Glycocalyx – Skin Flora Microscopic Examination It moves from skin surface (do not produce slime) to the Numerous gram-positive cocci catheter where it turns on slime production to firmly attach to the PMNs (Staphylococci are pus-formers) catheter. Once the slime is produced, it is impossible to get rid of it, Purulent exudates (fluid resulting from inflammatory processes), and the catheter usually has to be removed. joint fluids (expecting septic arthritis), and aspirated secretions Diagnosis Aspirate is best – maintain sterility w/ aseptic technique Colonies: Gray to white on primary isolation, small to medium- sized pin-heads, non-hemolytic (BAP) Coagulase-negative, (-) MSA Antimicrobial Test: Susceptible with 5 ug Novobiocin (16 mm to 27 mm Zone of Inhibition) Staphylococcus saprophyticus - CONS Normal flora: Female GUT, Perineum, and GIT Isolation of Staphylococci (ATCC 25923) UTIs (10-20%) in young sexually active women -> displacement Culture Media: BAP, MSA, PEA, CNA, CAP, BHI, Thioglycolate, of normal flora of vagina, perineum, and urethra and CHROM Agar Due in part to increased adherence to uroepithelium Grow easily on blood agar plates and thioglycolate o Causes Honeymoon Cystitis Heavily Contaminated: MSA and PEA Rarely present in other skin or mucous membranes Selective and Differential for MRSA: CHROM Agar Significant in urine culture even at low amount (10,000 CFU/mL) Isolation to suppress growth of non-staphylococcal and non- Common cause of UTI, 2nd to E. coli MRSA isolates and enhance isolation of the desired organism: Opportunistic pathogen and do not produce exotoxins High salt concentration (NaCl) or Antimicrobials (Cefoxitin) are Nosocomial infection: use of in-dwelling catheter added to culture media Increasing associated with opportunistic infection and not o After 24-48 hours of incubation: MRSA produces a regarded as harmless commensals (along with S. epidermidis) colored colony compared to colorless isolate of others Culture (BAP): White, some produce yellow pigments; Opaque, Thioglycolate slightly larger than pin-heads, non-hemolytic CoNS recovered from sterile sites and from sites associated with Biochemical Test: Coagulase Negative MSA indwelling devices should be considered potential pathogens Novobiocin Test: (5ug) Resistant (no zone of inhibition at 6 mm If heavily contaminated, use the following selective medium: to 12 mm) Mannitol Salt Agar Staphylococcus lugdunensis – CONS inhibits non-halophilic organisms, supports growth of halophiles Emerged as a virulent organism, has hemolysin and clumping ability of S. aureus to ferment mannitol and tolerate 10% NaCl factor and causing a disease spectrum like S. aureus; refractory Positive result: Golden yellow colonies treatment due to biofilm formation Phenol Red: pH indicator o More aggressive than the other CoNS in terms of o Fermentation of mannitol: Acidic (S. aureus) infectivity ▪ Phenol Red -> Yellow o Contains the mecA gene that codes for Oxacillin o No mannitol fermentation: Alkaline (other Staph spp.) resistance ▪ Phenol red -> Red/Pink Can be confused with S. aureus if the slide coagulase method is performed Columbia colistin-nalidixic acid agar (CNA) o CoNS by the tube coagulase method inhibits Gram-negative bacteria Infections: Infective Endocarditis, Meningitis, Septicemia, UTI, Phenylethyl Alcohol Agar Skin and Soft tissue infections selective media: adding inhibitory substances, desired organism Staphylococcus haemolyticus – CONS will grow and undesired organism will not grow) 2nd most common coagulase negative staphylococci S. aureus on BAP Found in wounds, UTIs, bacteremia, endocarditis o Recently noted resistance to vancomycin (MRSA) Golden yellow colonies, surrounded by a clear zone of hemolysis o Becomes significant due to isolation from blood culture (beta- hemolysis) especially in sheep or rabbit or rabbit blood agar in atmosphere or 20% CO2 Other Opportunistic Pathogens o S. lugdunensis S. aureus on NA o S. schleiferi Golden yellow and opaque colonies with smooth glistening