Gram-Positive Cocci PDF

Medical Bacteriology Gram positive cocci Staphylococcus Streptococcus Enterococcus  Staphylococcus spp. ▪ Staphylococcus is a genus of Gram-positive bacteria in the family Staphylococcaceae; currently consists of 49 species ▪ Gram-positive cocci (Ø...

Medical Bacteriology Gram positive cocci Staphylococcus Streptococcus Enterococcus  Staphylococcus spp. ▪ Staphylococcus is a genus of Gram-positive bacteria in the family Staphylococcaceae; currently consists of 49 species ▪ Gram-positive cocci (Ø  1 µm ) arranged in clusters ▪ Do not produce spores, not motile, exotoxin producing ▪ Staphylococci are ubiquitous in animals and human ▪ Survive on dry surfaces for long periods Staphylococcus aureus Staphylococcus epidermidis Staphylococcus saprophyticus Staphylococcus haemolyticus Staphylococcus lugdunensis Staphylococcus saprophyticus Staphylococcus spp.  Staphylococcus aureus ▪ Gram-positive cocci growing in clusters ▪ Capsulated ▪ Growth in aerobic/anaerobic conditions ▪ Growth with high concentration of salts S. aureus colonies can have a yellow or gold color as the result of the carotenoid pigments that form during their growth, hence the species name It produces coagulase. Most other staphylococcal species do not produce coagulase and are referred to collectively as “coagulase-negative staphylococci” (CoNS)  Epidemiology of S. aureus Humans are the reservoir of S. aureus: 15-50% of healthy individuals are carriers. S. aureus colonizes the skin and the nasopharynx, therefore it can be transferred to a susceptible person either through direct contact or through contact with fomites (e.g., contaminated clothing, bed linens) S. aureus: virulence factors  S. aureus: virulence factors MSCRAMM proteins (adhesins) (microbial surface components recognizing adhesive matrix molecules) = adhere to host matrix proteins (fibronectin binding protein, collagen binding adhesin) Polysaccharide capsule Protects the bacteria by inhibiting phagocytosis Protein A binds to the Fc region of IgGs preventing phagocytosis and inducing B-cell death → decreasing the production of antibodies specific for S. aureus IMMUNOGLOBULINS  S. aureus: secreted proteins involved in the pathogenesis ENZYMES TOXINS  Cytotoxins of S. aureus α - toxin ß – toxin or sphingomyelinase C disrupts the smooth muscle in blood vessels specific for sphingomyelin and and is toxic to many types of cells lysophosphatidylcholine; toxic for a variety of (erythrocytes, leukocytes, hepatocytes, and cells (erythrocytes, fibroblasts, leukocytes, platelets) and macrophages) δ - toxin γ – toxin wide spectrum of cytolytic activity (detergent- bicomponent toxin inducing cell lysis like action), affecting erythrocytes, many mediated by pore formation, with subsequent other mammalian cells, and intracellular increased permeability to cations and membrane structures osmotic instability PV (Pantom-Valentine leukocidin) toxin bicomponent toxin acting on leukocytes by pore formation  Superantigens of S. aureus Exfoliative toxin A: protease that splits the intercellular bridges in the stratum granulosum epidermis → skin exfoliation Enterotoxin: the precise mechanism of activity is not understood→stimulating intestinal peristalsis, nausea and vomiting (food poisoning) Toxic shock syndrome toxin-1 (TSST-1): massive release of cytokines by both macrophages (IL-1β and TNF-α) and T cells (IL-2, IFN-γ and TNF-β) → Superantigen acts as a bridge between clinically devastating toxic shock antigen presenting cells and T-cells inducing syndrome the release of large amounts of cytokine  Pathogenesis of S. aureus S. aureus causes disease through production of toxins or through direct invasion and destruction of tissue o The clinical manifestations of some staphylococcal diseases are almost exclusively the result of toxin activity (e.g., staphylococcal scalded skin syndrome, staphylococcal food poisoning, and toxic shock syndrome) o Other diseases result from proliferation of the bacteria, and the recall of PMNLs leading to abscess formation and tissue destruction (e.g., cutaneous infections, endocarditis, pneumonia, osteomyelitis, septic arthritis) →suppurative infections Staphylococcal Scalded Skin Syndrome (SSSS) and bullous impetigo SSSS and bullous impetigo (localized form of SSSS) are mediated by exfoliative toxins, proteases that split desmoglein-1(Dsg-1) responsible for the intercellular bridges in the granulosum epidermis. Normal skin SSSS: disseminated desquamation of epithelium in infants; blisters with no bacteria or leukocytes Bullous impetigo: localized cutaneous infection characterized by vesicle on an erythematous base  Enterotoxins and food poisoning Numerous distinct staphylococcal enterotoxins have been identified Enterotoxins are stable to heating at 100°C for 30 minutes and resistant to hydrolysis by gastric enzymes. o food can be contaminated with enterotoxin-producing staphylococci o once the toxins have been produced, neither mild re-heating of the food nor exposure to gastric acids will be protective o after consumption of food contaminated with enterotoxin: rapid onset of vomiting, diarrhea, and abdominal cramping, with resolution within 24 hrs Common contaminated food: processed meats such as ham and salted pork, custard- filled pastries Staphylococcal enterotoxins (SE)  Toxic Shock Syndrome Toxin-1 (TSST-1) Strains producing TSST-1 live mostly in the vagina of infected women: TSST-1 penetrates mucosal barriers while bacterium remains localized in the vagina (or at the site of infection as wounds) and it is responsible for the systemic effects of TSS (Toxic Shock Syndrome).→ hypotension and shock  Toxic Shock Syndrome (TSS) o The first reports of TSS were published in the 1980 in menstruating women. o These reports were followed by a dramatic increase in the incidence of TSS in women (and men). o Subsequently, it was discovered that TSST-1–producing strains of S. aureus could multiply rapidly in tampons and release toxin o After the recall of these tampons, the incidence of disease—particularly in menstruating women—decreased rapidly o Rare cases of TSS are associated with wounds o High mortality: death in patients with TSS is caused by: hypovolemic shock leading to multiorgan failure, multisystem intoxication characterized initially by fever, hypotension, and diffuse peeling, large skin lesions  Suppurative Infections Furuncles or boils: painful, Osteomyelitis: destruction of pus-filled cutaneous nodules bones Carbuncles: coalescence of furuncles with extension into subcutaneous tissues Septic arthritis (joint infection): painful erythematous joint with collection of purulent material in the joint space  Bacteremia and endocarditis S. aureus is a common cause of bacteremia and endocarditis Hematogenous pneumonia is common for patients with bacteremia  Laboratory diagnosis DIRECT DIAGNOSIS ONLY Biological samples: swabs, pus, blood, sputum, bronchoalveolar lavage Microscopic examination following GRAM stain (only if sampling from sterile sites) Culture on agar plate is the GOLD STANDARD Staphylococci grow rapidly on nonselective media incubated aerobically or anaerobically, with large, smooth colonies seen within 24 hours S. aureus colonies will gradually turn yellow  Laboratory diagnosis Culture on mannitol-salt agar plate S. aureus can be isolated selectively on mannitol-salt agar which is supplemented with mannitol (fermented by S. aureus but not by most other staphylococci) and 7.5% sodium chloride (inhibits the growth of most other organisms) Biochemical identification o Coagulase test o Catalase test o DNase test  MRSA strains Bacterial resistance to methicillin and related penicillins (methicillin resistant S. aureus, MRSA strains) is mediated by acquisition of a gene (mecA) that codes for a novel penicillin-binding protein - PBP2a, that has a low affinity for methicillin and related penicillins and cephalosporins Currently, the majority of S. aureus responsible for hospital- and community-acquired infections are resistant to these semisynthetic penicillins, and these MRSA strains are resistant to all β-lactam antibiotics - so-called “superbugs” The antibiotic that remained active against staphylococci is vancomycin Unfortunately, isolates of S. aureus have now been found with resistance to vancomycin (VRSA strains)  Coagulase-negative Staphylococci (CoNS) Staphylococcus epidermidis Staphylococci Staphylococcus saprophyticus are ubiquitous. All Staphylococcus haemolyticus persons have CoNS on Staphylococcus lugdunensis their skin Staphylococcus saprophyticus CoNS can infect prosthetic heart valves and, less commonly, native heart valves (endocarditis) A persistent bacteremia is generally observed in patients with infections of shunts and catheters (intravascular catheter associated infection)  STAPHYLOCOCCUS EPIDERMIDIS o Gram positive o Aero-anaerobic facultative, organized in clusters o Optimal growth temperature of 30-37°C o Found at the level of the stratum corneum and in the epidermal basement membrane in dry, moist and sebaceous regions  MUTUALISTICALLY SYMBIOTIC RELATIONSHIP BETWEEN SKIN AND MICROBES The skin hosts S. epidermidis and C. acnes and supplements the bacteria with nutrients, while in turn both bacteria partecipate in skin homeostasis, host defense and innate immunity. Dysbiosis is a disequilibrium of microbiota diversity and functionality, often characterized by an “abnormality” in bacterial composition, abundance or deficiency, and correlated to the development of inflammatory skin diseases according to the severity of the dysbiosis. The link between cause and consequence between microbiota dysbiosis and skin pathogenesis still remains unclear and is not well established to date S. epidermidis (CoNS) in biofilm formation  Streptococcus spp. ▪ Streptococcus is a genus of Gram-positive bacteria in the family Streptococcaceae; currently consists of > 100 species ▪ Gram-positive cocci arranged in pairs or chains ▪ Fastidious growth requirements, facultative anaerobes, catalase negative ▪ Do not produce spores, not motile, exotoxin producing Streptococcus pyogenes Streptococcus agalactiae Streptococcus mitis Streptococcus oralis Streptococcus mutans Streptococcus pneumoniae Streptococcus spp.  Streptococcus spp. Hemolytic patterns (α, β, γ) 1. the β-hemolytic streptococci, which are classified by Lancefield grouping - including S. pyogenes and S. agalactiae 2. the α-hemolytic (viridans streptococci) and γ- hemolytic streptococci, which are classified by biochemical testings – including S. mitis, S. pneumoniae, S. oralis Lancefield groupings Serological classification (originally A to W) based on group-specific cell wall antigens, most of which are carbohydrates Biochemical/physiologic properties  β-hemolytic streptococci Streptococcus pyogenes (group A) Streptococcus agalactiae (group B) typically appears as small colonies with typically appears as large colonies with a large zone of hemolysis small zone of hemolysis α- hemolytic streptococci Streptococcus mutans Streptococcus mitis partial hemolysis Streptococcus oralis Streptococcus pneumoniae  Streptococcus pyogenes Gram positive cocci, β-hemolytic pattern, Lancefield grouping A ANTIGENIC STRUCTURES o Peptidoglycan o Group-specific carbohydrate o Type-specific antigen, M protein o M-like surface proteins o Lipoteichoic acid o F protein o some strains: hyaluronic acid capsule (→molecular mimicry) Adhesion to host cells Immune evasion  Streptococcus pyogenes TOXINS Streptolysin S (SLS)→oxygen-stable o hemolysin capable of lysing erythrocytes, leukocytes, and platelets o stimulate the release of lysosomal contents after engulfment, with subsequent death of the phagocytic cell Streptolysin O (SLO)→oxygen-labile o Hemolysin (membrane-damaging protein) capable of lysing erythrocytes, leukocytes, platelets, and cultured cells o antibodies are formed against SLO (antistreptolysin O [ASO] antibodies), useful for documenting recent group A streptococcal infection Streptococcal pyrogenic exotoxins (SPE)→heat-labile (SpeA, SpeB, SpeC and SpeF) o act as superantigens, interacting with both macrophages and T cells, with the enhanced release of proinflammatory cytokines o responsible for necrotizing fasciitis, streptococcal toxic shock syndrome and scarlet fever  Streptococcus pyogenes ENZYMES Streptokinase (A and B) o break up blood clots and fibrin deposits so that the bacterium can spread from the initial site of infection. o encoded by phages DNases (A to D) o depolymeration of free deoxyribonucleic acid present in the infectious sites, reducing environmental viscosity Hyaluronidase o hydrolyzes hyaluronic acids in connective tissue promoting bacterial spread C5-peptidase o protects bacteria from clearance inhibiting a protein of the complement system  Streptococcus pyogenes Clinical diseases No disease: Transient colonization →asymptomatic infection →immunity Diseases: Suppurative diseases Non suppurative diseases  Streptococcus pyogenes Pharyngitis is primarily a disease of children (5-15 yrs), but infants and adults are also susceptible o S. pyogenes is spread from person to person through respiratory droplets o syntoms appear 2-4 days after exposure to the pathogen with abrupt onset of sore throat, fever, malaise, and headache o the posterior pharynx appears erythematous with an exudate o prominent cervical lymphadenopathy Scarlet fever is a complication of pharyngitis that occurs when the infecting strain is lysogenized by a bacteriophage SPE-producer o within 1 to 2 days after the onset of the pharyngitis, a diffuse erythematous rash appears on the upper chest and to the extremities o a yellowish-white coating initially covers the tongue → protrusion of the red papillae through the white coating gives the tongue a "white strawberry" appearance  Streptococcus pyogenes Soft-tissue infections are preceded by skin colonization with streptococci, after which the organisms are introduced into the superficial or deep tissues through a break in the skin Pyoderma (impetigo) is a confined, purulent infection of the skin that primarily affects exposed areas (face, arms) Erysipelas is an infection of the skin. Patients experience localized pain, erythema, warmth, lymph node enlargement, and systemic signs (chills, fever) Streptococcus pyogenes “Flesh-eating bacteria” Necrotizing fasciitis and necrotizing myositis = infection that occurs deep in the subcutaneous tissue, spreads along the fascial planes, and is characterized by an extensive destruction of muscle and fat → development of toxic shock syndrome  Streptococcus pyogenes Streptococcal toxic shock syndrome (STSS) is a rare, but serious bacterial infection. STSS can develop very quickly into low blood pressure, multiple organ failure, and even death. o the strains mainly involved are M1 and M3 serotypes, they have a prominent hyaluronic acid capsule and produce SpeA and SpeC o patients initially experience soft-tissue inflammation at the site of infection, pain, fever, malaise, vomiting and diarrhea. o the pain intensifies to shock and organ failure  Streptococcus pyogenes Rheumatic fever or Rheumatic heart disease (RHD) is a nonsuppurative complication of streptococcal pharyngitis o rheumatogenic strains (M1 serotype) induce a vigorous antibody response in patients with pharyngitis → cross-reactive antibody deposition in the heart, joints, skin with inflammation o involvement of the heart manifests as endocarditis, pericarditis, myocarditis Incidence is reduced with antibiotic treatment of pharyngitis  Streptococcus pyogenes Acute Glomerulonephritis is a nonsuppurative sequela of both pharyngeal and pyodermal streptococcal infections o specific nephritogenic strains of group A streptococci are associated with the disease o immune complex deposition in the kidney → acute inflammation of the renal glomeruli with hypertension, hematuria, and proteinuria  Laboratory diagnosis MICROSCOPY ▪ Gram staining, observation of Gram positive cocci in pairs or chains associated with leukocytes ▪ Biological specimes: throat swabs (in case of pharingytis); swab of lesions with purulent material; surgically removed purulent material (in case of cellulitis or fasciitis), blood (in case of suspected systemic infection) ANTIGEN DETECTION Immunologic tests using antibodies that react with the group-specific carbohydrate in the cell wall can be used to detect group A streptococci directly in throat swabs  Laboratory diagnosis CULTURE GOLD STANDARD for identification of S. pyogenes is bacterial growth on blood agar plates, with observation of β-hemolytic colonies IDENTIFICATION Bacitracin susceptibility test S. agalactiae resistant to bacitracin S. pyogenes susceptible to bacitracin  Laboratory diagnosis ANTIBODY DETECTION Detection in the serum sample of antibodies against streptolysin O (ASO test) These antibodies appear 3 to 4 weeks after infection and their detection is useful for confirming rheumatic fever or acute glomerulonephritis, resulting from a recent streptococcal pharyngeal infection ASO PRINCIPLE  Treatment of S. pyogenes infection S. pyogenes is very sensitive to penicillin, so oral penicillin V or amoxicillin can be used to treat streptococcal pharyngitis For penicillin-allergic patients, an oral cephalosporin or macrolide may be used. Antibiotic therapy in patients with pharyngitis speeds the relief of symptoms and, if initiated within 10 days of the initial clinical disease, prevents rheumatic fever. Patients with a history of rheumatic fever require long- term antibiotic prophylaxis (IM penicillin G) to prevent recurrence of the disease. Because damage to the heart valve predisposes these patients to endocarditis, they also require antibiotic prophylaxis before they undergo procedures that can induce transient bacteremias (dental procedures).  Streptococcus agalactiae Gram positive cocci, β-hemolytic pattern, Lancefield grouping B o Gram positive bacterium arranged in pairs or chains (= S. pyogenes) o Large colonies with a narrow zone of β-hemolysis (≠ S. pyogenes) o Virulence factor: polysaccharide capsule with antiphagocytic properties (group B antigen) It is one of the main agents of severe infection in the newborn, and transmission occurs from the mother to the newborn in utero (rare), at birth, as the 5-30% of women are colonized by S. agalactiae in the vagina and/or rectum.  Streptococcus agalactiae Neonatal infections present as two different clinical entities: o Early-onset disease, characterized by sepsis and pneumonia within the first 7 days of life o Late-onset disease with meningitis and sepsis between 7 days and 3 months of age → mortality rate: 1-6% In an effort to prevent neonatal disease, it is recommended that all pregnant women should be screened for S. agalactiae at 35 to 37 weeks’ gestation. Colonized women should undergo IntraVenous penicillin G or ampicillin, at least 4 hours before delivery, to reduce the risk of transmission. Infections of adult patients may be observed as postpartum infections or in immunocompromised subjects. The spectrum of infections includes pneumonia, bacteremia, endocarditis, urinary tract infections, skin and soft tissue infections.  Streptococcus pneumoniae Gram positive cocci, α/β-hemolytic pattern, NO Lancefield grouping S. pneumoniae is an encapsulated Gram positive, lancet shaped coccus Cells are oval and arranged in pairs (commonly referred to as diplococci). Colonies appear α-hemolytic on blood agar if grown aerobically, and may be β- hemolytic if grown anaerobically. Colonial morphology varies, with colonies of encapsulated strains generally large round, and mucoid, and colonies of nonencapsulated strains smaller and flat in blood agar. All colonies undergo autolysis with aging: the central portion of the colony dissolves, leaving a small depression in the center of the colony  Streptococcus pneumoniae STRUCTURE o Peptidoglycan o Two forms of teichoic acids: teichoic acid exposed to the cell surface (C polysaccharide), unrelated to Lancefield grouping carbohydrate, precipitate the C- reactive protein; teichoic acid bound to the lypid of cytoplasmic membrane (F antigen) o Phosphorylcoline is unique in this cell wall and is involved in cell lysis o Polysaccharide capsule: the major anti-phagocytic surface element of pneumococci and the major protective antigen; the surface capsular polysaccharide of S. pneumoniae provokes a type-specific protective immune response  Streptococcus pneumoniae S. pneumoniae is a human pathogen that colonizes the oropharynx and then, in specific conditions, is able to spread to the lungs, paranasal sinuses and middle ear, leading to pneumonia, sinusitis, otitis media or meningitis when the bacteria is spread by blood to the brain. Colonization of the throat and nasopharynx in healthy individuals is up to 62% (transient colonization with different serotypes starting from the 6 months of age) Colonization is mediated by: o surface protein adhesins: adherence to epithelial cells of the nasopharynx: does not usually produce a symptomatic infection o IgA-protease: destroy secretory IgA produced in the mucosa o Pneumolysin is a potent cytotoxin, which can form pores in membranes, thereby killing any cell; it promotes intra-alveolar replication of pneumococci, the penetration of pneumococci from the alveoli into the interstitium, and dissemination of the organisms into the bloodstream (INVASION)  Streptococcus pneumoniae S. pneumoniae is transmitted from asymptomatic carriers or patients with the disease through the aerosol. Bacteria adhere to the epithelial cells of the nasopharynx thereafter spread to the lungs (pneumonia), paranasal sinuses (sinusitis), ears (otitis media). Spread of S. pneumoniae in blood (bacteremia) can occur with all of these diseases (meningitis)  Pneumococcal pneumonia Pneumococcal meningitis It develops when the bacteria multiply in It occurs more frequently in the elderly; the alveolar spaces (lobar pneumonia). neurological sequelae, including hearing The onset of the clinical manifestations of loss, focal neurological deficits and cognitive pneumococcal pneumonia is abrupt, impairment are estimated to occur in 30 to consisting of a severe shaking chill and 52% of surviving patients. sustained fever of 39°C to 41°C Mortality from pneumococcal meningitis ranges from 16 to 37%.  Laboratory diagnosis MICROSCOPY o Gram staining, observation of diplococci Gram positive o Biological specimens: sputum, purulent material, cerebrospinal fluid (CSF), blood PNEUMOCOCCAL ANTIGEN DETECTION Immunologic tests able to detect the pneumococcal capsule polysaccharide that is present in CSF and in urine of patients NUCLEIC ACID–BASED TESTS PCR assays have been developed for identification of S. pneumoniae isolates in CSF CULTURE o from sputum, blood, CSF on blood agar plates. o S.pneumoniae is susceptible to the antibiotic optochin  Treatment of S. pneumoniae infection Penicillins (amoxicillin) (50% of strains are resistant) Macrolides (erythromycin), or tetracycline but resistance has also become common For serious pneumococcal infections, treatment with a combination of antibiotics is recommended until in vitro susceptibility results are available: Vancomycin combined with ceftriaxone is used commonly for empirical treatment, followed by monotherapy with an effective cephalosporin, fluoroquinolone, or vancomycin.  Streptococcus pneumoniae 13-valent conjugated pneumococcal vaccine o Recommended for infants younger than 2 years o 4 doses (at 2, 4, 6, and 12 to 15 months) are recommended 23-valent pneumococcal polysaccharide vaccine: o Recommended for children older than 2 years and adults (single dose)  Enterococcus The enterococci are Gram positive cocci, arranged in pairs or in short chains. They growth under several cultural conditions (aerobically/anaerobically, broad temperature and pH range, in the presence of NaCl and bile salts) They are enteric bacteria commonly recovered in feces from humans and animals. E. faecalis and E. faecium are found in the large intestine and in the genitourinary tract. They are nosocomial human pathogens: they are responsible for urinary infections (mainly associated with urinary catheterization or instrumentation. Endocarditis can be a severe complication. Broad spectrum of antibiotic--resistance  Laboratory diagnosis MICROSCOPY and CULTURE Gram staining Culture in blood agar Bile Esculin agar is a selective (→ 40% of bile salts) differential (→ esculin) medium Enterococcus hydrolyze esculin to glucose and esculetin, that in turns reacts with ferric ions, resulting in the blackening of the medium.