P B Valenzuela, MD, FPCPInternal Medicine – Infectious Diseases

Classify the gram-positive organisms and enumerate their member-species of medical importance

Describe the gram positive organisms by their growth, colonial and morphologic characteristics

Correlate clinical findings with the organisms’ antigenic and virulence determinants

Offer treatment options

The Gram-positive cocci◦ Staphylococci◦ Streptococci

The Gram-positive bacilli ◦ Spore-forming◦ Non-spore-forming

2 medically important genera◦ Staphylococci◦ Streptococci

Non-motile, non-spore-formers

2H202 2H2O + O2 Hydrogen Peroxide

Staphylococci: Catalase +Streptococci: Catalase -

Catalase bubbles

Gram-positive, spherical, in grape-like irregular clusters

Gray to golden-yellow colonies (S. aureus)

Gray to white (S. epid)

Staphylococcus aureus

Coagulase-negative staphylococci

Staphylococcus epidermidis

Staphylococcus saprophyticus

Gram Stain + coccus + coccus + coccus

Alpha Toxin + — —

Mannitol fermentation + — +/-

coagulase + — —

Novobiocin senstivity + + —

Cell wall◦ Peptidoglycan

Elicits production of interleukin -1, opsonic antibodies Chemoattractant for PMNs Activates complement

◦ Teichoic acids - antigenic◦ Protein A (in many S. aureus strains)

Binds to the Fc region of IgG Decreases the efficiency by which S aureus are

opsonized and phagocytosed

Capsule◦ Present in some strains◦ Inhibit phagocytosis by PMNs

Surface ProteinsAdhesins◦ Coagulase

Considered synonymous with invasive pathogenic potential Binds to prothrombin thrombin fibrinogen fibrin..... attaches to surface of staph Alters ingestion/destruction by phagocytic cells

◦ Clumping factors Bind to fibrinogen and fibrin Produces the typical clusters of staphylococci when mixed

with plasma Aid in adherence to traumatized skin

Toxins◦ α toxin: hemolysis of erythrocytes, necrosis of

skin, and release of cytokines and eicosanoids that may produce shock

◦ β toxin: sphingomyelinase; act on lipid membranes

◦ γ toxin: three proteins that interact with the two proteins of PVL --- form 6 toxins WBC lysis by causing pore formation in the cellular

membranes◦ δ toxin: disrupts biologic membranes

*All 4 are also called hemolysins

◦ Panton-Valentine Leukocidin (PVL) Synergistic with γ toxin Pore-forming cytotoxin; lysis of WBCs,

◦ Exfoliative toxins: dissolves the mucopolysaccharide matrix of the epidermis desquamation, seen in staphylococcal scalded skin syndrome (SSSS); promotes spread of staph under the statum corneum. Epidermolytic toxin A: chromosomal gene product;

heat stable Epidermolytic toxin B: plasmid-mediated; heat labile

Both are superantigens

◦ Toxic Shock Syndrome Toxin (TSST) In ~20% of S. aureus isolates Associated with fever, shock, multisystem involvement,

desquamation◦ Enterotoxins

~50% of S. aureus strains can produce >/= 1 enteroroxin Produced when S. aureus grows in CHO and CHON foods Heat –stable; resistant to gut enzymes Important cause of food poisoning – vomiting and diarrhea toxin acts on the gut neural receptors stimulation of

the vomiting center

Natural reservoir: Humans and other mammals

S. epidermidis: part of the normal flora of the skin, respiratory tract and the GI tract

S. saprophyticus: UTI in young women Colonization of the anterior nares with S.

aureus : 20-50%; also found on the axilla, perineum, vagina

Prototype lesion: furuncle/localized abscess

Coagulase fibrin formation of abscess wallAccumulation of inflammatory cellsLiquefaction of necrotic tissue in the center of lesionDrainage Slow filling of the cavity with granulation tissue

healing

Skin and soft tisssue infections Invasive infections Device-related infections

Toxin-mediated syndromes

Skin/Soft tissue Impetigo Cellulitis Abscess Bullous impetigo Wound infection

Disseminated Infection Endocarditis Osteomyelitis Pulmonary infections

Secondary localization Focal suppuration

Toxin-mediated◦ Toxic Shock Syndrome

Abrupt onset of fever, vomiting, diarrhea, myalgia, rash, hypotension

Multiorgan failure Background of infected wound, tampon use, localized infections

◦ SSSS fever widespread tender erythema that quickly forms thin-walled,

fluid-containing bullae that rupture, leaving a moist base of skin

◦ Food poisoning Short incubation period: 1-8 hours No fever Nausea, vomiting, diarrhea Rapid recovery

Drainage◦ For abscesses

Antimicrobial therapy◦ S. aureus

90 % Penicillin-resistant 35% methicillin-resistant; correlates with (+) mecA

Mueller-hinton agar with 6 ug/mL oxacillin/ commercial assay/ PCR

DOC: Beta lactamase resistant penicillins: Nafcillin/methicillin/ Oxacillin/Dicloxacillin/ Cloxacillin

IV antibiotics for severe infections Vancomycin, for MRSA Linezolid, Daptomycin, quinopristin-dalfopristin

Antimicrobial therapy◦ S. epidermidis

75% are resistant to nafcillin/oxa/methicillin DOC: Vancomycin

◦ S. saprophyticus DOC: Amoxicillin-clavulanic acid, cephalosporins

2H202 2H2O + O2 Hydrogen Peroxide

Staphylococci: Catalase +Streptococci: Catalase –

Note: Nonstreptococcal Catalase-negative Gram-positive cocci and coccobacilli (Aerococcus, Gemella, Leuconostoc, Pediococcus, Lactobacillus)

Catalase

bubbles

Gram +, spherical, in pairs and chains

Gram stain of sputum (X1000)

Colony morphology and hemolytic reactions Serologic specificity of the cell wall

◦ group-specific substance (Lancefiled classification)◦ Other cell wall and capsular antigens

Biochemical reactions◦ Sugar fermentation reactions; tests for the

presence of enzymes; susceptibility ◦ Used for species that typically do not react with

antibody tests for the group-specific substances Ecologic features

Beta-hemolysis: complete lysis of RBCs; clear zone around colonies

Alpha-hemolysis: incomplete lysis of RBCs; green/brownish zone

Gamma-hemolysis: no hemolysis

Basis of serologic grouping: A-H, K-U Group specific carbohydrate determined by an

amino sugar:◦ Group A rhamnose-N-acetylglucosamine◦ Group B rhamnose polysacharide◦ Group C rhamnose-N-acetylgalactosamine◦ Group D glycerol teichoic acid containing D-alanine and glucose◦ Group F glucopyranosyl-N-

acetylgalactosamine◦ Group G◦ Non- groupable

Groupable Groups A, B, D – frequent Groups C,G, F – less frequent

Non-Groupable S. pneumoniae Viridans streptococci

Hemolytic reactions Hemolytic reactions Groups A and B Groups A and B

◦β Group D Group D

◦ α or or γ S. pneumoniaeS. pneumoniae and viridans and viridans

◦α

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Name Lancefield Group

Hemolysis Other features

S . pyogenes A Beta Colonies >0.5 mmBacitracin susceptible;

PYR+

S. agalactiae B Beta CAMP +, Na hippurate +

Bacitracin-resistant

E. faecalis D Alpha, Gamma

(V)

Bile-esculin +; grow on 6.5% NaCl

S. bovis D Alpha, Gamma

(V)

Bile-esculin +; no growth in 6.5% agar

S. pneumoniae

NA Alpha Optochin Susceptible

S. viridans NA Alpha, Gamma

Optochin resistant

S. anginosus group (milleri group)

F (A,C,or G)

Beta, Alpha,Gamma

Small colonies (< 0.5 mm); Grp A

Bacitracin-resistant, PYR -

Peptostreptococcus

NA Alpha, Gamma

Obligate anaerobes

Org Habitat Mode of transmission

S pyogenes Skin, upper respi tract Direct contact, droplets

S agalactiae Female genital tract, lower GIT

Acquired by neonates in utero or during delivery

S pneumo Nasopharynx Contaminated secretions

Viridans strep Oral cavity Usually endogenous strains gaining access to normally sterile sites; dental manipulations

Enterococcus Soil, food, waterGIT, female GUT

Usually endogenous strains gaining access to normally sterile sites; nosocomia

Virulence factors Lipoteichoic acid

◦ fimbriae◦ binds to epithelial cells

Capsule ◦Hyaluronic acid

M protein ◦ antigenic◦ anti-phagocytic

Toxins and enzymes

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fibronectinfibronectin

lipoteichoic acidlipoteichoic acidF-proteinF-protein

epithelial cellsepithelial cells

Toxins and enzymes

Suppurative◦ Wound Infections, with spread to tissues/bloodstream

Erysipelas Cellulitis Necrotizing fasciitis; “flesh-eating bacteria” Puerperal fever Bacteremia/sepsis

◦ Local infection Pharyngitis –most common infection due to S. pyogenes Pyoderma

◦ Invasive Streptococcal Toxic Shock Syndrome Scarlet Fever

Non-Suppurative or Poststreptococcal diseases

Post-streptococcal Glomerulonephritis 3 wks after skin infection; <0.5% incidence Majority recover completely

◦ AGN CGN Kidney failure Nephritogenic strains

◦ M type 12, 4 , 2, 49, 59-61 Antigen-antibody complexes Hematuria, proteinuria, edema,

hypertension Low complement levels

Rheumatic Fever most serious sequela of S. pyogenes;

important cause of valvular heart dse in the young

Preceded by infection 1-4 weeks earlier◦ Higher incidence with patients with severe

pharyngitis Cross reaction of M-protein with myosin Reactivated by recurrent strep infections

(GN does not) need for prophylaxis

Streptococcus Disease entity Tx

Group B – S. agalactiae

Neonates: meningitis, RDS, sepsis

Pen, ampi

Group D - Enterococci UTI, wound, biliary tract/abdominal, blood infections, endocarditis; Important nosocomial pathogen

Ampi, Pen, +aminoglycosides (synergistic)Intrinsically resistant to cephalosporins, penicillinase resistant pen (i.e. cloxacillin), monobactams

- S. bovis Endocarditis, bacteremia in the background of colon cancer

Pen, ampi

Viridans streptococcus

Endocarditis Pen

S. pneumoniae Pneumonia, endocarditis

Pen

Bacillus sp Clostridium sp

Square ends Long chains Central location of spores

Aerobic Saprophytic; soil, water, air,

vegetation; from carcasses of dead animals

Gray to white colonies B. anthracis: (-) hemolysis,

non- motile other bacilli: (+) hemolysis,

motile

B cereus

B anthracis

Virulence◦ Capsule

Determined by the capsule gene on the plasmid Poly-D-glutamic acid; Antiphagocytic

◦ Anthrax Toxin Toxin genes on plasmid Protective antigen (PA) – binds to receptors

facilitate entry of EF and LF Edema factor (EF)- adenylyl cyclase; together with

PA forms edema toxin Lethal factor (LF) – Together with PA forms lethal

toxin

Injured skin (Cutaneous anthrax; 95%)

Ingestion mucous membranes (Gastrointestinal anthrax; rare)

Inhalation (Pulmonary anthrax; 5%)

Sepsis can occur

Localized and systemic infection: endocarditis, meningitis, pneumonia, osteomyelitis

Eye infections: associated with trauma Device-related infections

Toxins – produced during sporulation or during log-phase growth

Emetic form◦ fried rice, pasta◦ Incubation of 1-5 hours◦ Nausea, vomiting, abdominal cramps,

occasionally diarrhrea◦ Self-limiting

Diarrheal type◦ meat dishes and sauces◦ Incubation of 1-24 hours◦ Profuse diarhea

≥ 105 per gram of food = diagnostic May be present in normal stool specimen

Spores◦ Wider than the bacilli◦ Central/subterminal or

terminal

Colony◦ Anaerobic conditions◦ C. pefringens : large, raised◦ C. tetani : small, raised

Soil, animal feces Wound infections C botulinum toxin

Toxin types A-GReleased during growth and during autolysis◦ Toxin type A and E – cleave SNAP 25◦ Toxin type B cleaves synptobrevinProteolysis of SNARE proteins in neurons inhibition

of acetylcholine release at the synapse paralysis

◦ Destroyed by heating x 20 mins, 100 deg C.

SNARE proteins

• Spiced/smoked/vacuum packed/ canned alkaline food eaten without cooking; honey

• Incubation: 18-24 hrs• Visual disturbances, swallowing dificulty, speech

difficulty, respiratory paralysis

• Dx: demonstration of toxin in the serum/leftover food. Radioimmunoassay Passive hemeagglutination

◦ Tx: supportive, ventilatory support : antitoxin (trivalent)

Infection – strictly localized Toxin Tetanus

◦ Incubation: 4-5 days, up to several weeks◦ Tetanospasmin

Binds to receptors on the presynaptic membranes of motor neurons migrate to spinal cord, brain via retrogarade axonal transport blocks release of glycine and GABA (inhibitory) hyperrefelexia, spasms

◦ Spasms of ms in the area of injury, trismus, lockjaw, respiratory compromise .... Death

◦ Dx: clinical; anaerobic culture◦ Tx: wound debridement, anti toxin, toxoid, antibiotic

(pen G/ Metronidazole)

Typically do not form spores when grown on laboratory media

Food Poisoning – meat dishes Enterotoxin – in some strains; producing

diarrhea; nausea/vomiting less frequent; self-limiting

Invasive◦ Toxins and enzymes: hemolytic and nectrotizing

effects Alpha toxin (lecithinase): Splits lecithin (component of cell wall)

into phosphorylcholine and diglyceride Theta toxin Dnase Hyaluronidase: digests collagen of subcutaneous tissue and

muscle

Contamination from soil/feces or from GIT Vegetative cells multiply, ferment CHO

+gas tissue distention+ compromised blood supply

+ toxins, enzymes Rapid spread; crepitation in the subcutaneous

tissue/muscle, foul smelling discharge, fever, shock... Death

Tx: early surgery, antibiotic Tx, antitoxins

In gas gangrene (clostridial myonecrosis)◦ Usually mixed infection: + g + cocci, g- bacilli

Spread of infection

Background of ◦ antibiotic therapy (commonly clindamycin,

ampicillin)◦ diarrhea (bloody or watery)◦ Fever, abdominal cramps, leukocytosis

Dx Detection of toxin/s

Toxin A (entrotoxin) Toxin B (cytotoxin)

Endoscopic findings od pseudomembranes Microabscesses in the bowelTx D/C antibiotic/s Oral Vancomycin or Metronidazole

Corynebacterium Propionibacterium Listeria Erysipelothrix Actinomycetes

Coryneforms: Short, slightly curved rods, rounded ends

Matachromatic granules – beaded appearance

Aerobic Small, granular, gray,

irregular borders on Blood agar

Enhanced growth on Loeffler medium

Pleomorphic morphology

4 biotypes: gravis, mitis, intermedius, belfanti

Respiratory tract, skin, wounds Can produce toxin Diphtheria Toxin

◦ Absorbed into the mucus membranes necrosis of epithelium and superficial inflammation pseudomembrane Fragment A : abrupt arrest of protein synthesis in the

epithelial cells Fragment B : required for the transport of fragment A

into the cell◦ Also produces paralysis

Sore throat Fever Dyspnea Difficulties with vision,

speech, swallowing, movement of limbs

Heart involvement Attempt to remove the

pseudomembrane bleeding

Dx◦ Modified Elek method

(WHO Diphtheria reference Unit)

◦ PCR◦ ELISA◦ Immunochromographic

strip Tx

◦ Antitoxin◦ Antibiotics

(Penicillin/erythromycin)

Elek method

Anaerobic corynebacteria P acnes is aerotolerant Part of normal flora of skin Common contaminant in blood culture

Produces lipases split free fatty acids off from skin lipids inflamacne

Occasionally, device related infections

Short rods, singles or in short chains

Facultative anaerobe +hemolytic motile Enhanced isolation if

tissue is kept at 4 deg C for a few days

Listeria monocytogenes motility test in semisolid medium showing the typical "umbrella" growth

Entry thru the GIT; ingestion of contaminated dairy products, meat, vegetables

Internalin (cell wall protein) interacts with E cadherin receptor on epithelial cells promotes phagocytosis into epithelial cells

Listeriolysin O Toxin - lyses membrane of phagolysosome org escapes into cytoplasm proliferate, cell actin proliferation induced by actA (a cell wall protein) propelled to form filopods

Filopods are ingested by adjacent cells organism is released new cycle

Iron

Perinatal◦ Early-onset (granulomatosis infantiseptica): result

of infection in utero; sepsis, pustular lesions and granulomas in multiple organs

◦ Late-onset: meningitis between birth and 3rd week Adult

◦ Bacteremia◦ Meningoencephalitis◦ Focal infections (rare)Non-specific clinical presentation

Dx◦ Culture of blood and CSF

Tx◦ Ampicillin◦ Erythromycin◦ Cotrimoxazole

Either ◦ short rods ◦ Small, smooth coloniesOR◦ long filaments◦ Large, rough colonies

May show alpha hemolysis

Direct inoculation 2-7 days incubation Pain and swelling at the site of

infection Resolve in 3-4 weeks

Tx: PenG

Actinomyces Nocardia Rhodococcus Streptomyces Actinomadura

Bacilli with tendency to form chains/filaments

Bcilli, coccobacillary, branching filaments

Cell wall contain mycolic acid- Partially acid-fast

Extensive branching, with aerial filaments; fragment after formation

Mostly, an opportunistic infection Sub acute to chronic pulmonary infection; may

disseminate to brain/skin Fever, weight loss, chest pain

Dx◦ Culture, biopsy

Tx◦ Cotrimoxazole

/amikacin/imipenem/minocycline/linezolid/cefotaxime

Mycetoma (‘Madura foot’)◦ Localized, slowly progressive chronic infection

that begins in the subcutaneous tissue◦ Destructive◦ Painless◦ May be fungal

Actinomycetoma◦ Nocardia asteroides, N brasiliensis/ Streptomyces

somaliensis / Actinomadura madura◦ Tx: Cotrimoxazole, streptomycin, dapsone

A israelii, A naeslundii, others Part of Normal oral flora Facultative anaerobes Heaped up “molar tooth” colonies Yellowish sulfur granules in infected tissue

Initiated by trauma Chronic, suppurative and granulomatous

infection that produces pyogenic lesions with interconnecting sinus tracts

Contains granules, containing microcolonies of orgs

Concomittant bacteria present Commonly affects

◦ Cervicofacial area◦ Thoracic◦ abdominal

Dx◦ Exam for the presence of sulfur granules

Tx◦ Penicillin/erythromycin