LKCR 3BMT 13

Bacteriology - Preliminary Coverage

Points of genera differentiation Micrococci Microscopic appearance Staphylococci Gram (+) cocci singly, in pairs and in clusters Nonmotile Non spore forming Aerobic/Facultati ve anaerobic *except S. saccharolyticus = obligate anaerobe + depends on organism
*S. aureus, S.

Streptococci Gram (+) cocci in pairs or chains Nonmotile Non spore forming Facultative anaerobic / aerotolerant anaerobes -

Enterococci

Neisseria Gram (-) diplococci

*N.elongata and N. weaveri = rod shaped

Gram (+) cocci

Gram (+) cocci

Motility Spore-Forming

Nonmotile Non spore forming

depends on specie Non spore forming Facultative anaerobic Pseudocatalase (Weak bubbling)

Nonmotile Non spore forming

Atmospheric Requirement

Strict aerobic

Aerobic

+
*N. elongata = catalase negative

Catalase Test

Coagulase Test

intermedius, S. schleiferi, S. lugdunensis = (+) contains Staphylocoagulase

Colony appearance

With yellow pigment in BAP

Round, Medium sized, cream-colored, white or rarely light gold and "buttery-looking"

Oxidase/ Bacitracin Susceptibility

+ (Sensitive)

(Resistant)

Small and somewhat transparent *Hemolysis depends on organism + (susceptible) : S.pyogenes and S. pneomoniae only

Usually non-hemolytic

Depends on organism

(Resistant)

Oxidase (+)

LKCR 3BMT 13

Bacteriology - Preliminary Coverage

Laboratory Diagnosis of Staphylococcus sp. S. aureus Pinhead, white, creamy, -hemolytic Yellow colonies in MSA (fermenta tion of mannitol) + S. epidermidis S. saprophyticus S. haemolyticus S. ludgunensis S. schleiferi

Cultural Characteristics in BAP

Pinhead, non-hemolytic white to gray colonies

Slightly larger colonies with about 50% of the strains producing yellow pigmentation

Medium sized with moderate or weak hemolysis and variable pigmentation

Medium sized and hemolytic

No description found

Coagulase test Clumping factor Pyrrolidonyl Arylamidase Voge-Proska uer (VP) test
Novobiocin susceptibility

+ + +
(Susceptible)

+ +

+ on slide test - on tube test Some strains (+) + + +

(Susceptible)

+ on slide test - on tube test Some strains (+) + + +

(Susceptible)

+ (Susceptible)

(Resistant)

+ (Susceptible)

Virulence Factors of Staphylococcus sp. Organism Virulence Factor Enterotoxin A, B, D Enterotoxin B,C > G,I Enterotoxin B Toxic Shock Syndrome Toxin-1 / Enterotoxin F Exfoliative Toxin / Epidermolytic toxin -Hemolysin -Hemolysin (sphingomyelinase C) Pathogenicity Food poisoning Toxic Shock Syndrome Staphylococcal pseudomembranous enterocolitis Menstruating-associated TSS Staphylococcal Skin Scalded Syndrome / Ritter disease Damage to platelets and macrophages and cause severe tissue damage Hot-cold lysin ; enhances hemolytic activity at 37C & 4C

Staphylococcus aureus

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Bacteriology - Preliminary Coverage

Staphylococcus epidermidis

Staphylococcus saprophyticus

Staphylococcus lugdunensis

Suppresses phagocytosis and associated with severe cutaneous - Hemolysin associated with infections and necrotizing Panton-Valentine leukocidin (PVL) / pneumonia Staphylococcal leukocidin Associated with community acquired staphylococcal infections Hydrolyzes hyaluronic acid in Hyaluronidase connective tissues, permitting spread of bacteria Ability to bind the Fc portion of Protein A immunoglobulin G (IgG) -lactamase Resistance to penicillin Nosocomial/Hospital-acquired UTIs Poly-gamma-DL-glutamic acid and (indwelling catheters, prosthetic Biofilm production devices) Slime Infective endomielitis UTIs in young women (adheres more effectively to the lining of the epithelial cells in UG tract) Oxacillin Resistance Gene mecA More virulent and mimics S. aureus infections

Infections caused by S. aureus Folliculitis- relatively mild infection of a hair follicle or oil gland Furuncles- boils; large, raised superficial abscesses Carbuncles- larger, more invasive lesions develop from multiple furuncles, which may progress into deeper tissues Bullous impetigo- larger and surrounded by a small zone of erythema; highly contagious Scalded Skin Syndrome (SSS)/Ritter Disease- extensive exfoliative dermatitis Toxic Shock Syndrome (TSS)- associated with highly absorbent tampon use; two types (menstruatingand non-menstruating-associated) Toxic Epidermal Necrolysis (TEN)- most commonly drug-induced due to hypersensitivity reaction Food poisoning- ingestion of contaminated food (enterotoxins do not cause any detectable odor or change in the appearance or taste of the food) Pneumonia- contagious, lower respiratory tract infection; multiple abscesses and focal lesions in the pulmonary parenchyma Osteomyelitis- bacteria gains entrance into the blood and may lodge in the diaphysis of the long bones MRSA- Methicillin-Resistant Staphylococcus Aureus MRSE- Methicillin-Resistant Staphylococcus Epidermidis Vancomycin- drug of choice for serious staphylococcal infections VISA- Vancomycin-Intermediate S. Aureus

LKCR 3BMT 13

Bacteriology - Preliminary Coverage

VRSA- Vancomycin-Resistant S. Aureus D-test- Double Disk Diffusion test (Macrolide resistance: erythromycin and clindamycin) Classification of Streptococcus and Enterococcus Species Lancefield Group Antigen Hemolysis Type(s) Common Terms Disease Association(s)
Rheumatic fever, scarlet fever, pharyngitis, glomerulonephritis, pyogenic infections

S. pyogenes

Group A Strep

S. agalactiae

Group B Strep

S. dysgalactiae, S. equi

Group C Strep Non-enterococcus (cannot grow on 6.5% NaCl), member of viridans strep Enterococcus

Neonatal Sepsis, meningitis, puerperal fever, pyogenic infections Pharyngitis, impetigo, pyogenic infections Endocarditis, UTIs, pyogenic infections UTIs, pyogenic infections Pneumonia, meningitis, pyogenic infections Pyogenic infections, endocarditis, dental caries, abscesses in various tissues

S. Bovis

, none

E. faecalis, E. faecium S. pneumoniae

, , none

--

Pneumococcus

Anginosus group, mutans group, mitis group, salivarus group

A, C, F, G, N

, , none

Viridans Strep

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Bacteriology - Preliminary Coverage

Types of Hemolysis Hemolysis Alpha () Beta () Non hemolytic Alpha-prime () or wide zone Description Partial lysis of RBCs around colony Greenish discoloration of area around colony Complete lysis of RBCs around colony Clear area around colony No lysis of RBCs around the colony No change in agar Small area of intact RBCs around colony surrounded by a wider zone of complete hemolysis

Laboratory Diagnosis of Streptococcus sp. S. pyogenes Lancefield Antigen Hemolysis A S. agalactiae B

Grayish, white, mucoid colonies surrounded by a small zone of -hemolysis S. dysgalactiae Non-entero cocci Enterococci
S. pneumoniae

Viridans Strep A,C,F,G,N , , none

D , none

D , , none

, none

Colonial characteristics on BAP

Small, transparent & smooth with a welldefined area of -hemolysis

Small Pyogenic colonies, streptococci usually large colony non-hemol ytic

Small colonies with occasional , hemolysis

Large zone of -hemolysis Young colonies hemolysis have a w/ green round, discoloratio glistening, n, fastidious wet, mucoid, domeshape

PYR hydrolysis Hippurate hydrolysis CAMP hydrolysis

LAP (Leucine aminopeptidase) hydrolysis

+ +

+ + +

+ +

+ +

VP test
-D-Glucuronidase

+/S

-/R

+ -/R

-/R

-/R

+/S

Mitis group= (-)

-/R

Bacitracin Susceptibility

LKCR 3BMT 13

Bacteriology - Preliminary Coverage

Vancomycin Susceptibility Optochin Susceptibility Bile Esculin Growth in 6.5% NaCl Bile Solubility

+/S -/R -

+/S -/R -

+/S -/R -

+/S -/R + -

S>R -/R + + -

+/S +/S +

+/S -/R -

Antigenic Structure and Virulence Factors of Streptococcus sp. Organism Antigenic structure Virulence factor M protein encoded by emm genes Fibronectin-binding protein (protein F) and Lipoteichoic acid Hyaluronic acid capsule Streptococcus pyogenes M protein Streptolysin O (SLO) Pathogenicity Resistance to phagocytosis and adherence to mucosal cells Adhesion molecules that secure attachment Prevents opsonized phagocytosis and masks its antigen to remain undetected Oxygen-labile; highly immunogenic, lyses leukocytes, platelets, etc. Oxygen-stable; lyses leukocytes and non-immunogenic Lysis of fibrin clots Separates tissue and spreads infection Scarlet fever Prevents phagocytosis but is ineffective after opsonization; most significant component: Sialic acid Inhibits activation of the alternative pathway of complement system Protection

Streptolysin S Streptokinase Hyaluronidase (Spreading factor) Streptococcal pyrogenic exotoxins (Spes)

Streptococcus agalactiae

Group B-specific antigen (acid-stable polysaccharide)

Capsule

Sialic Acid Streptococcus C-substance that reacts Capsular polyssaccharide

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Bacteriology - Preliminary Coverage

pneumoniae

with C-reactive protein Extracellular dextran and adhesins Adherence and colonization in endocarditis Similar to group A

Viridans Streptococci

Polysaccharide capsule and cytolysin

Enterococcus

Not stated

Protein M, streptolysin O, hyaluronidase, DNAse, Streptokinase Extracellular surface protein, extracellular serine protease, gelatinase Cytolysin

Colonization and adherence to heart valves Two-subunit toxin similar to bacteriocins

Clinical Infections of Streptococci sp. Organism Clinical Infection Pharyngitis Strep throat Impetigo Pathology/Definition Most common clinical manifestation of group A strep infection Most common in children (5-15 yo); sore throat, malaise, fever Localized skin disease, from small vesicles to weeping lesions Acute spreading skin lesion that is intensely erythematous with a plainly demarcated but irregular edge Deeper invasion of streptococci Diffuse red rash that appears on upper chest and spreads to trunk and upper extremities, followed by desquamation Invasive infection; rapidly progressing inflammation and necrosis of the skin, subcutaneous fat and fascia; flesh-eating disease Entire organ system shuts down, leading to death Rheumatic fever and acute glomerulonephritis (AGN) MOT: Vertical transmission of the organism from the mother to

Erysipelas Cellulitis S. pyogenes Scarlet fever

Necrotizing fasciitis

Streptococcal Toxic shock syndrome Post-streptococcal sequelae S. agalactiae Invasive disease of the newborn

LKCR 3BMT 13

Bacteriology - Preliminary Coverage

Bacterial pneumonia S. pneumoniae Pneumococcal pneumonia Empyema Subacute bacterial endocarditis Oral infections (since they are oropharyngeal commensals) UTIs and bacteremia

newborn Infections often presents as Pneumonia or meningitis with bacteria Number one cause of pneumonia in elderly patients and patients with underlying diseases Sudden onset of chills, dyspnea and cough Pleural effusion Most common infection Gingivitis and dental caries Most common

Viridans Streptococci (Opportunistic pathogens) Enterococcus

Laboratory Diagnosis of Pathogenic Neisseria sp. and Moraxella catarrhalis N. gonorrhoeae Small, grayish-white, translucent, raised with entire edge Urethra (Male) Endocervix (Female) porB + + + (immediate, vigorous bubbling) + + N. meningitidis Slightly larger than N. gonorrhoeae, bluish-gray in BAP or small, tan, sometimes mucoid, convex colonies on CAP Nasopharynx, oropharynx, urogenital and rectal sites porA and porB + + (weak, delayed bubbling) + + + M. catarrhalis Smooth, opaque, gray to white, hockey puck colonies

Colonial Morphology

Primary Isolation Sites Presence of Por Catalase (3% H2O2) test Oxidase test Superoxol (30% H2O2) test Growth on Modified Thayer Martin, Martin Lewis, NY City agar Growth on Nutrient medium (BAP, CAP) at RT Acid production from Glucose Acid production from Maltose

Upper respiratory tract None + + -

Some are (+), some are (-) + -

LKCR 3BMT 13

Bacteriology - Preliminary Coverage

Acid production from DNase Reduction of Nitrate & Nitrite

Production of Hydroxyprolylaminopeptidase

+ + -

Virulence Factors of N. gonorrhoeae and N. meningitidis Organism Virulence factor Receptors for human transferrin Capsule, cell membrane proteins I, II, and III, and Lipooligosaccharide (LOS) endotoxin Pili (fimbriae) Pathogenicity Compete with the human hosts for Iron Protection and antigenic variation Initial attachment to host tissues *T1, T2= Possess pili, virulent forms *T3-T5= devoid of pili, avirulent, found in subcultures Forms channels for nutrients to pass into and waste products to exit the cell Facilitate adherence Blocks host serum bactericidal (IgG) against the organism Same pathogenicity Pandemic United States (B= community-acquired disease) Meningococcal pneumonia Invasive diseases

N. gonorrhoeae

Porin protein (Por) or protein I Protein II (Opa/Opacity) Protein III (Rmp / Reduction modified protein) Same virulence factors as N. gonorrhoeae Group A strains N. meningitidis Serogroup B and C Serogroup Y Serogroup W-135

Clinical infections of N. gonorrhoeae, N. menigitidis, M. catarrhalis Organism Clinical Infection Acute urethritis, purulent discharge, dysuria, AHU strains isolated Pelvic inflammatory disease (PID), sterility, ectopic pregnancy, perihepatitis (Fitz-Hugh-Curtis syndrome) Pathology/Diagnosis Found in men

N. gonorrhoeae

Found in women

LKCR 3BMT 13

Bacteriology - Preliminary Coverage

10

Ophthalmia neonatorum

Epidemic meningitis

Fulminant meningococcemia or sepsis N. meningitidis Hemorrhage Bacteremia Waterhouse-Friderichsen syndrome Upper respiratory tract infection Lower respiratory infections

Gonococcal eye infection in newborns MOT: Infected birth canal in vaginal delivery *erythromycin= antimicrobial eyedrop Onset of frontal headache, stiff neck, sometimes fever May occur with or without meningitis once N. meningitidis enters bloodstream; with DIC, septic shock and Waterhouse-friedrichsen syndrome Pupura with petechial skin rash Tachycardia and hypotension may develop Hemorrhage in the adrenal glands In healthy children and elderly In adults with Chronic obstructive pulmonary disease

M.catarrhalis

Laboratory Diagnosis of Non-pathogenic Neisseria sp.

N. flavescens

N.cinerea

N. lactamica

N. mucosa

N. polysacc -harea +

N. sicca

N. subflava

N. elongata

N. weaveri

Growth on selective media Growth in BAP, CAP Carbohy drate metaboli sm Catalase test Oxidase test
Reduction of Nitrate

+ + -

+ + -

+ + -

+ + +

+ + -

+ + -

+ + -

+ -

+ + -

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Bacteriology - Preliminary Coverage

11

Reduction of Nitrite

+ Small, grayish, white, translusc ent

*Point of differentiati on w/ N.
gonorrhoeae = N.cinerea

varied Small, grayish white with yellow ring (only one to use lactose)

varied

Varied

+
Rod-shaped in miscrope Large, grayish white with yellowish tinge

Colonial morphology

Golden yellow colonies in BAP or CAP

Large, often adherent to the agar and very mucoid

Small, gray (sometms yellowish), translusce nt, raised

Dry, wrinkled adherent, and breadcrumb like *sicca= dry

*subflava = less yellow

Small, semi-op aque with a smooth appeara nce

is +/S with Colistin

Selective Media for the Isolation of N. gonorrhoeae and N. meningitidis Selective Medium Thayer-Martin Inhibitory Agents Vancomycin Colistin Nystatin Vancomycin Colistin Nystatin Trimethoprim Vancomycin Colistin Anisomycin Trimethoprim Vancomycin Colistin Amphotericin B Trimethoprim Vancomycin Lincomycin Colistin Amphotericin B Trimethoprim Suppressed Organisms Gram (+) Gram (-) Yeast Gram (+) Gram (-) Yeast Swarming Proteus spp. Gram (+) Gram (-) Yeast Swarming Proteus spp. Gram (+) Gram (-) Yeast Swarming Proteus spp. Gram (+) Gram (+) Gram (-) Yeast Swarming Proteus spp. Capnocytophaga sp.

Modified Thayer-Martin

Martin-Lewis

New York City

GC-LECT