Gram-positive: Staphylococci and Streptococci Medical Microbiology SBM 2044

Staphylococcus aureus The most common pus-forming (pyogenic) bacteria Can produce focal abscess, from the skin (furuncles, boils) to the lungs, osteomyelitis, kidneys and endocarditis Include S. aureus, S. epidermidis, S. saprohyticus (UTI) S. aureus can persist in the body because they have numerous cell surface virulence, exotoxins and enzymes

S. aureus Staphylo = grape clusters (Greek) A large 1µm Gram-positive coccus growing in cluster-of-grapes shape. Can survive in long period of dry, on inanimate object. Heat resistant. Identification: large, creamy colonies on nutrient agar; catalase +

Staphylococci

S. aureus Encounter Major reservoirs = humans Live on skin – grow at high salt and lipid concentrations because they make lipases and glycerol ester hydrolases, that degrade skin lipids Colonise skin and mucosal surfaces using MSCRAMMs: –Fibronectin–binding proteins (FnbpA, FnbpB) –Collagen-binding –Clumping factors A and B Spread person-to-person by direct contact or airborne

S. aureus pathogenesis Entry Tissue penetration upon skin or mucosal membrane damaged by cut Spread and Multiplication Survival in tissues dependon – no. of entering microorganisms – site involved – speed of body’s inflammatory responses – immunological history of the host

Damage Local infections  pus collection, i.e. abscess Staphylococci can spread into subcutaneous and submucosal tissues and caused cellulitis Activate acute inflammatory reaction, pouring in chemotactic factors Damaged area are usually localised by the formation of thick-walled fibrin capsule : center of abscess is necrotic with debris of dead cells Why many virulence factors? S. aureus pathogenesis

Surface structures: Capsules – inhibit phagocytosis Peptidoglycan – interacts with TLR-2, activate alternative pathway Teichoic acid – C’ activation and adherence to mucosal cells Protein A – binds to Fc terminus of IgG Secreted factors: Catalase – H 2 O 2  H 2 O Coagulases – fibrinogen  fibrin Pore-forming toxins – create channels to disturb cellular homeostasis Haemolysins – Leukocidin Hyaluronidase – hydrolyse matrix of connective tissues Β-lactamase – hydrolyse penicillin Penicillin-binding protein (PBP2a)

S. epidermidis Normal flora, rarely caused disease Infections of S. epidermidis with other catalase-negative staphylococci in patients implanted with artificial devices e.g. prosthetic joints or IV catheters Results in septicaemia and endocarditis Possibly peptidoglycan or slime layer allows the organisms to stick to the surface of plastics

S. saprophyticus Caused cystitis in young women

Staphylococcal toxin diseases Staphylococcal scalded skin syndrome (SSSS) –Exfoliative toxins A and B – highly tissue specificserine proteases that causes separation of the layers of the epidermis at the desmosomes Staphylococcal toxic shock syndrome (TSS) – characterised by fever, skin rash, hypotension, peeling of the skin – use tampons – oxygenated vagina and stimulate toxin production –TSST-1, staphylococcal enterotoxins A  E Virulence gene regulation – two-component regulatory systems –Accessory gene regulator (Agr), staphylococcal respiratory response (Srr)

Diagnosis Gram stain and culture Treatment Methicillin-sensitive S. aureus – Rx: semi- synthetic penicillins and cephalosporins Methicillin-resistant S. aureus – Rx: vancomycin vancomycin-resistant S. aureus –acquired the genes of resistance from vancomycin-resistant Enterococcus species

Streptococci Classification –Haemolytic pattern – in blood agar media, colonies formed may be surrounded by a clear zone of haemolysis (α,β, γ) –Group-specific antigens (Lancefield classification) – by serological reactivity of extracted cell wall antigens (A  U) –Species – biochemical tests

Streptococcus  Gram-positive  Grow in chains  Non-motile  Facultative anaerobes  Early studies distinguished 3 broad groups on blood agar non-haemolytic  -haemolytic  -haemolytic  Homofermentative [Glucoselactic acid]

Streptococci

 GAS diseases – changing patterns Changes in virulence of prevailing GAS strains ? Changes in social conditions – less crowding? Changes in herd immunity to prevailing virulent strains?  Reemergence of severe invasive infections Sporadic cases since mid-1980s – new virulent strains? Streptococcal toxic shock o Some cases associated with obviously severe tissue infections o Many others – shock following mild or unapparent infections Sporadic – implies predisposing factors

Group A Streptococci Epidemiology –Ubiquitous worldwide –Common diseases are acute pharyngitis or pyoderma infections (skin and soft tissues) Encounter –Carriers appear asymptomatic –Person-to-person spread is mediated by respiratory droplets or by direct contact to skin Entry –For pyodermal infections, streptococci need to gain entry into deeper layers of skin –In pharyngeal infections, to prevent from being swept away, GAS must lipoteichoic acid (LTA),protein F and M protein

Spread and multiplication –Most GAS remain localised to the site of initial infection –In pharynx and tonsils, may result in erythema and exudate associated with strep throat –Peritonsillar abscess (quinsy) or spread to adjacent structures (mastoid and middle ear) –Impetigo in skin –Erysipelas and cellulitis in deeper layers of skin –GAS may spread laterally in deep tissues, by secreting enzymes necrotizing fasciitis and myositis

Secreted proteinFunction Proteases Hyaluronidasespreading factor Streptolysins S and Oform pores in the host membranes Deoxyribonucleases (DNase) Streptokinase– bind to human protein plasminogen, and convert this to plasmin plasmin degrades fibrin

GAS virulence factors – Excreted products  Cytolytic toxins Streptolyxin O (SLO) Streptolysin S (SLS) Produced by all strains Both lyse wide range of cells, including PMNs suppuration and/or necrosis Various subtle effects at sub-lytic concentrations  -haemolysis SLO – ‘sensitive’ to O 2 SLS – stable in O 2 “Thiol-activated” toxin (NB: Salyers & Whitt misleading - neither are “enzymes” )

WT sloΔ1 sagBΔ1 sloΔ1- sagBΔ1 PBS Weight gain (grams) ~3 x 10 9 cfu ~3 x 10 8 cfu ~3 x 10 7 cfu ~3 x 10 5 cfu ~3 x 10 6 cfu Sterile SLO- and SLS-defective mutants  murine s.c. model - weight loss at 24h post infection

M protein Important for cell adherence to keratinocytes Prevent opsonization by complement – bind to fibrinogen and interferes with the alternative pathway – bind with host complement control proteins and inhibit opsonins formation Hypervariable regions of M protein are antigenic, but there are > 100 M protein serotypes

Hyaluronic acid capsule Antiphagocytic structure on bacterial surface Hyaluronic acid is abundant in human connective tissue - hence GAS can camouflaged themselves But capsule may interfere with the adherence of GAS to epithelial cells – so GAS shed the capsule during the early stages of infection using hyaluronidase

Damage GAS can evoke an intense inflammatory responses in tissues Streptococcal pyrogenic exotoxins (SPE) –SPE A, B and C cause rash, a characteristic of scarlet fever –SPE A and C are bacterial superantigens that activate a large subset of T cells Immunologically mediated disease (nonsuppurative sequelae) –acute rheumatic fever (ARF) –acute post-streptococcal glomerulonephritis

Diagnosis Impetigo – a cluster of small vesicles on a pink base that breaks down to honey- coloured crusts Erysipelas – a raised, bright red patch of skin More difficult to diagnose streptococcal pharyngitis – rapid strep tests – throat cultures (throat swab)

Treatment and Prevention Penicillin – 10 day oral therapy Erythromycin or other macrolide antibiotics for individuals allergic to penicillin

> 100 distinct M types of GAS distinguished since then - called M1, M2, M3, M4,……..etc. M-type specific antigen was sensitive to proteases M proteins Streptococcus pyogenes  Natural habitat: Humans  Strains distinguished by M serotyping Devised by Lancefield in 1920s, using panels of absorbed sera to hot-HCl extracted antigen, she called “M antigen”  Highly versatile pathogen Toxinogenic diseases Suppurative infections Immunologically-mediated diseases

Group A Streptococci Principle sites of infection: Pharyngitis, tonsillitis, otitis media, sinusitis. Local spread (e.g.) Invasive infections Bacteraemia or septicaemia Deep-seated tissues Skinpyroderma, erysipelas Pharynx Streptococcal Toxic Shock Occasionally Extensive necrosis (necrotizing fasciitis) Other tissues Puerperal fever (childbirth fever) in women – major killer in past

Follicular tonsillitis Tonsillitis Streptococcus pyogenes

Cellulitis Erysipleas Streptococcus pyogenes Impetigo

(< 24 hours post surgery) Streptococcus pyogenes Necrotizing fasciitis

Streptococcus pyogenes

Scarlet fever Streptococcus pyogenes

Toxic Shock  In past – probably linked to scarlet fever  Since mid-1980s – associated with ‘new’ highly virulent strains - rapidly fulminating some cases in previously healthy young adults, no obvious predisposing factors  Associated with production of superantigenic toxins, but other factors also involved

rheumatic heart disease (RHD) (ca 50% cases) - damage to heart valves, permanent scaring in survivors antibodies and/or T cells X-react with host antigens? (later) Acute rheumatic fever (ARF)  autoimmune disease - triggered only by GAS pharyngitis  associated with strong immune response to GAS.  Symptoms arise > 10days + after GAS infection responsible GAS strain already ‘cleared’  Symptoms may include: inflammation multiple sites, starting with major joints (arthritis) neurological disorders (Sydenhams chorea)

Acute rheumatic fever (ARF)  Initial attack rates low (3% in untreated pharyngitis) Widespread prescription of penicillin for ‘sore throats’ Remarkably, GAS have not (yet ?) developed resistance to penicillins  High recurrence (up to 50%) - increasing in severity

Post-streptococcal acute glomerulonephritis (PSGN): GAS infections - complications  Common, but rarely life-treating - some GAS infections of either pharynx or skin.  Symptoms arise some 10 days after infection Most probably entrapment of GAS antigen-host antibody complexes at basement membranes of glomeruli reflect kidney dysfunction, probably involving inflammation of glomeruli  Pathogenesis: might also involve an ‘autoimmune’ response

Mesangial cell intrusion Endothelial cell, has Basement membrane Small complexes diffuse thro’ basement membrane into urine, but the occasional larger complex can’t & is normally removed by mesangial cell Normal glomerulus Glomerulonephritis Inflammation PMN 100 nm pores Too much large immune complex entrapment ? S. pyogenes infections Example: Sequel of some

Group B streptococci Streptococcus agalactiae are aerobic G+ diplococci that are β-haemolytic on blood agar plates – found in lower GIT and female genital tracts – GBS is a leading cause of neonatal sepsis and meningitis – prevent opsonization and phagocytosis with a polysaccharide capsule

Enterococci Enterococcus faecalis cause UTI, wound infections, endocarditis, intraabdominal abscesses and bacteremia. –Normal flora of GIT and GUT – resistant to bile and high salt concentrations – nosocomial infections – resistance to many antibiotics, often bacteriostatic – bacterial killing must use a combination treatment of a β-lactam and an aminoglycoside