Staphylococcus and Related Organisms

미생물학교실 권 형 주

STAPHYLOCOCCISTAPHYLOCOCCI• Gram positive• Facultative anaerobes (Capable of aerobic and anaerobic growth)

• Grape like-clusters• Catalase positive• Major components

of normal flora - skin- Nose : mucose membrane

Gram stain of Gram stain of Staphylococcus aureusStaphylococcus aureus

1. 병원성 Staphylococcus 의 특징을 이용 하여 다른 세균과 구별

* 병원성 : 혈액응고효소 생산 , 만니톨 분해 , 젤라틴 액화 , 황금색 집락 , 간혹 용혈성

식중독 , 화농성 염증 ,포도구균성 피부박탈증후군 , 독소형 쇼크증후군

Micrococcus : 그람양성구균으로 불규칙한 배열을 하고있으며 , 임상적으로 큰 의의는 없어 균속만 동정 : Catalase +, Coagulase -

Staphylococcus Catalase 시험 : catalase 생산세균을 동정 .(2H2O2

---catalase--- 2H2O + O2 )

양성 -Micrococcus, Staphylococcus. Bacillus, 장내세균 .

음성 -Streptococcus, 혐기성균 .

색소 생산시험 : 세균이 배지에 집락을 형성할때 색소를 생산

병원성균일 경우 황금색 색소 생성

S. aureus - 황색 S. epidermidis -( 회 ) 백색 ~ 레몬색 S. saprophyticus - 백색

Coagulase 생산시험 : 강한병원성인 Staphylococcus aureus 와 S. intermedius 및 S. saprophyicus 의 일부가 생산하는 효소이며 , 토끼나 사람의 혈장을 배양균과함께 반응시키면 혈장 의 응고가 일어남 .

* free coagulase- 혈장중의 prothrombin 을 활성화함으로써 응고

* bound coagulase( 균에 결합 )-fibrinogen 을 직접 fibrin 으로 바꿈Streptococcus용혈성 관찰 : 세균의 적혈구 파괴를 확인하기위해 혈액한천배지를 이용한다 .

배양후 집락주위에 형성된 용혈환에 따라 - 용혈로 구분

- 용혈 : hemoglobin 을 methemoglobin 으로 변화시켜 녹색의 환을 형성

- 용혈 : 적혈구가 완전히 파괴되어 투명한 용혈환 형성

- 용혈 : 비용혈성

Bacitracin 감수성 검사 : bacitracin 에 감수성이 있는 균은 13mm 이상 저지대 형성

Organism Historical Derivation

Staphylococcus staphylé, bunch of grapes; coccus, grain or berry (grapelike coccus)

S. aureus aureus, golden (golden or yellow)

S. epidermidis epidermidis, outer skin (of the epidermis or outer skin)

S. lugdunensis Lugdunum, Latin name for Lyon, France, where the organism was first isolated

S. saprophyticus sapros, putrid; phyton, plant (saprophytic or growing on dead tissues)

BOX 21-1. Important Staphylococci

Organism Diseases

Staphylococcus aureus

Toxin mediated (food poisoning, scalded skin syndrome, toxic shock syndrome); cutaneous (carbuncles, folliculitis, furuncles, impetigo, wound infections); other (bacteremia, empyema, endocarditis, osteomyelitis, pneumonia, septic arthritis)

Staphylococcus epidermidis

Bacteremia; endocarditis; surgical wounds; urinary tract infections; opportunistic infections of catheters, shunts, prosthetic devices, and peritoneal dialysates

Staphylococcus saprophyticus

Urinary tract infections; opportunistic infections

Staphylococcus lugdunensis

Arthritis, bacteremia, endocarditis, opportunistic infections, and urinary tract infections

Staphylococcus haemolyticus

Bacteremia, bone and joint infections, endocarditis, urinary tract infections, wound infections, and opportunistic infections

Table 21-1. Staphylococcus Species and Their Diseases

Physiology and Structure

O CAPSULE AND SLIME LAYER - polysaccharide capsule - inhibiting phagocytosis of the organisms by polymorphonuclear leukocytes

(PMN). - A loose-bound, water-soluble film (slime layer) : monosaccharides, proteins, and small peptides : Bind the bacteria to tissues and foreign bodies : important for the survival of relatively avirulent coagulase-negative staphylococci.

O PEPTIDOGLYCAN- layers of glycan chains built with 10 to 12 alternating subunits of N-acetylmuramic acid and N-acetylglucosamine

- endotoxin-like activity, stimulating the production of endogenous pyrogens, activation of complement, production of interleukin-1 from monocytes, and aggregation of PMN

O Synthesis of cell wall peptidoglycan

- Transpeptidation : cross-linking peptidoglycan

- Penicillin-binding proteins (PBPs)

: Targets of penicillin and beta-lactam antibiotics

- Methicillin-resistant S. aureus (MRSA)

: mecA gene (Staphylococcal cassette chromosome mec(SCCmec)

- novel penicillin-binding protein (PBP2)

- not bound by penicillin, retain enzymatic activity

- Hospital, community infections

- SCCmec type IV : most common type

o Methicillin-resistant Staphylococcus aureus (MRSA) - a bacterium responsible for difficult-to-treat infections in humans. - multiply-resistant Staphylococcus aureus or oxacillin-resistant Staphylococcus aureus (ORSA). - Community-Associated MRSA (CA-MRSA) or Hospital-Associated MRSA (HA-MRSA) - Survive treatment with beta-lactam antibiotics, (penicillin, methicillin, and cephalosporins)

Electron micrograph of MRSA

O TEICHOIC ACIDS - Ribitol teichoic acid with N-acetylglucosamine residues ("polysaccharide A") is present in S. aureus- Glycerol teichoic acid with glucosyl residues ("polysaccharide B") is present in S. epidermidis. - Attachment of staphylococci to mucosal surfaces through their specific binding to fibronectin. - poor immunogens, a specific antibody response is stimulated

O PROTEIN A - surface of most S. aureus strains - a unique affinity for binding to the Fc receptor of immunoglobulin (Ig)G1, IgG2, and IgG4

: prevents antibody-mediated immune clearance of the organism. - Extracellular protein A can also bind antibodies : consumption of the complement : a specific identification test for S. aureus.

O COAGULASE AND OTHER SURFACE ADHESIN PROTEINS

- clumping factor (bound coagulase) : outer surface of most strains of S. aureus : binds fibrinogen and converts it to insoluble fibrin, causing the staphylococci to clump or aggregate. - Other surface proteins : MSCRAMM (microbial surface components recognizing adhesive matrix molecules) proteins : adherence to host matrix proteins, which in turn bind to host tissues (e.g., fibronectin, fibrinogen, elastin, collagen).

Pathogenesis and Immunity - surface proteins : adherence of the bacteria to host tissues - extracellular proteins, such as specific toxins and hydrolytic enzymes.- expression of the exoprotein genes is controlled primarily by a global regulator, agr, which in turn is controlled by environmental factors, cell density, and energy availability.

STAPHYLOCOCCAL TOXINS O Cytotoxin 1) Alpha (α) toxin : bacterial chromosome and a plasmid : disrupts the smooth muscle in blood vessels and is toxic to many types of cells, including erythrocytes, leukocytes, hepatocytes, and platelets : integrated in the hydrophobic regions of host cell membrane, leading to formation of 1- to 2-nm pores. - rapid efflux of K+ and influx of Na+, Ca2+, and other small molecules leads to osmotic swelling and cell lysis.

2) Beta (β) toxin-sphingomyelinase C : toxic to a variety of cells, including erythrocytes, fibroblasts, leukocytes, and macrophages : hydrolysis of membrane phospholipids in susceptible cells

3) Delta (δ) toxin :a wide spectrum of cytolytic activity, affecting erythrocytes, many other mammalian cells, and intracellular membrane structures - nonspecific membrane toxicity : acts as a surfactant disrupting cellular membranes by means of a detergent-like action.

O Cytotoxin 4) Gamma (γ) toxin (made by almost all S. aureus strains) and P-V leukocidin (made by <5% of S. aureus strains) : Cell lysis by these toxins is mediated by pore formation with subsequent increased permeability to cations and osmotic instability.

o Panton-Valentine leukocidin (PVL) - cytotoxin—one of the pore forming toxins. - increased virulence of certain strains (isolates) of Staphylococcus aureus. : Methicillin-resistant Staphylococcus aureus (MRSA) - The cause of necrotic lesions involving the skin or mucosa, including necrotic hemorrhagic pneumonia. - The genetic material of a bacteriophage which infects Staphylococcus aureus, making it more virulent.

Mechanism of action- secrete lethal factors :secrete two proteins—toxins designated LukS-PV and LukF-PV, 33 and 34 kDa in size.- induces pores in the membranes of cells : assembling in the membrane of host defense cells, particularly white blood cells, monocytes and macrophages. The subunits fit together and form a ring with a central pore through which cell contents leak and which acts as a superantigen.

O Exfoliative Toxins ( 표피탈락독소 )

- Staphylococcal scalded skin syndrome (SSSS, 포도알균 열상피부증후군 ) : exfoliative dermatitis 원인 : serine proteases - splitting of the intercellular bridges (desmosomes) in the stratum granulosum epidermis : SSSS is seen mostly in young children and only rarely in older children and adults- ETA (heat stable, chromosome), ETB (heat labile, plasmid)

O Enterotoxins( 장독소 ) - stable to heating at 100°C for 30 minutes - resistant to hydrolysis by gastric and jejunal enzymes. - precise mechanism of toxin activity is not understood - superantigen ; releases inflammatory mediators- staphylococcal food poisoning.

O Toxic Shock Syndrome Toxin-1 ( 독소충격증후군 유발독소 -1)

- pyrogenic exotoxin C and enterotoxin F- superantigen that stimulates release of cytokines, producing leakage of endothelial cells at low concentrations and a cytotoxic effect to the cells at high concentrations. - ability of TSST-1 to penetrate mucosal barriers- Death in patients with TSS is cause by hypovolemic shock( 저혈액량쇼크 )- multiorgan failure.

O Superantigens- Exfoliative toxin A- Enterotoxin- TST-1

STAPHYLOCOCCAL ENZYMES O Coagulase - S. aureus strains possess two forms of coagulase: bound and free. - Bound : convert fibrinogen to insoluble fibrin and cause the staphylococci to clump. - Cell-free coagulase : formation of coagulase-reacting factor to form staphylothrombin, a thrombinlike factor. : conversion of fibrinogen to insoluble fibrin : protecting the organisms from phagocytosisO Catalase - catalyzes the conversion of hydrogen peroxide to water and oxygen

O Hyaluronidase Hyaluronidase hydrolyzes hyaluronic acids, the acidic mucopolysaccharides present in the cellular matrix of connective tissue - facilitates the spread of S. aureus in tissues.

O Fibrinolysin Fibrinolysin, also called staphylokinase : dissolve fibrin clots.

O Lipases - hydrolyze lipids, an essential function to ensure the survival of staphylococci in the sebaceous areas of the body. to invade cutaneous and subcutaneous tissues and for superficial skin infections

O Nuclease A thermostable nuclease - role of this enzyme in the pathogenesis of infection is unknown.

O Penicillinase - More than 90% of staphylococcal isolates were susceptible to penicillin in 1941- Resistance to penicillin quickly developed - produce penicillinase (β-lactamase) - transmissible plasmids.

Epidemiology

- Normal flora on human skin and mucosal surfaces – 피부 , 구인두 , 위장관 , 비뇨생식계

- Organisms can survive on dry surfaces for long periods (owing to thickened peptidoglycan layer and absence of outer membrane-characteristics of all gram-positive bacteria)

- Person-to-person spread through direct contact or exposure to contaminated fomites (e.g., bed linens, clothing)

- Risk factors include presence of a foreign body (e.g., splinter, suture, prosthesis, catheter), previous surgical procedure, and use of antibiotics that suppress the normal microbial flora

- Patients at risk for specific diseases include infants (scalded skin syndrome), young children with poor personal hygiene (impetigo and other cutaneous infections), menstruating women (toxic shock syndrome), patients with intravascular catheters (bacteremia and endocarditis) or shunts (meningitis), and patients with compromised pulmonary function or an antecedent viral respiratory infection (pneumonia) – 신생아간호에 중요

- Infections found worldwide and generally with no seasonal prevalence (except that food poisoning is more common in summer and during late-year holidays)

항생제 내성과 치사율

Community-associated MRSA (CA-MRSA)

Staphylococcus aureus

Clinical Diseases

One of commonest opportunistic One of commonest opportunistic infections - hospital and communityinfections - hospital and community:

• Toxin activity : SSSS : Staphylococcal food poisoning : Toxic shock syndrome

• Proliferation (abscess formation, tissue destruction) : cutaneous infections, endocarditis, pneumonia, empyema, osteomyelitis, septic arthritis

O Staphylococcal Scalded Skin Syndrome

( 포도알균 열상피부증후군 , 표피탈락증후군 )

- In 1878, Gottfried Ritter von Rittershain : bullous exfoliative dermatitis ( 297 infants younger than 1-month old)

- Ritter's disease or SSSS : perioral erythema (redness and inflammation around the mouth)

- Slight pressure displaces the skin (a positive Nikolsky's sign)--> large bullae or cutaneous blisters desquamation of the epithelium- bacterial toxin (Exfoliative Toxins) , protective antibodies appear- 전신성 표피탈락성 피부염이 가장 심각

O Bullous impetigo ( 수포성농가진 )- 표피탈가성 환부가 있는 국소적 질환- specific strains of toxin-producing S. aureus (e.g., phage type 71) - formation of superficial skin blisters. - Nikolsky's sign is not present. The disease occurs primarily in infants and young children and is highly communicable.

: A skin condition in which the top layers of the skin slip away from the lower layers when slightly rubbed

- Nikolsky's sign

: positive or negative. - A positive : loose skin that slips free from the underlying layers when rubbed. : The area beneath is pink and moist and may be very tender. : by twisting a pencil eraser against your skin. If positive, a blister will form in the area, usually within minutes.

- Allergic reaction (Toxic epidermal necrolysis) - Autoimmune condition (Pemphigus vulgaris) - Bacterial infection ( Scalded skin syndrome)

Staphylococcal Food poisoning• not an infectionnot an infection• food contaminated by humans food contaminated by humans

– growth of bacteriagrowth of bacteria– production of enterotoxinproduction of enterotoxin– enterotoxins are heat-stable

• onset and recovery both occur within few hoursonset and recovery both occur within few hours• Vomiting• nausea• diarrhea• abdominal pain• Enterocolitis –watery diarrhea, abdominal cramps, fever

Toxic shock syndrome (Toxic shock syndrome ( 독소충격증후군독소충격증후군 ))• FeverFever

• HypotensionHypotension

• rashrash

• desquamationdesquamation

• vomitingvomiting

• diarrheadiarrhea• Toxic shock toxin Toxic shock toxin

- DisseminationDissemination• OrganismOrganism

– no disseminationno dissemination- ability of TSST-1 to penetrate mucosal barriers- Death in patients with TSS is cause by hypovolemic shock( 저혈액량쇼크 )- multiorgan failure.

Cutaneous Infections

Localized, pyogenic staphylococcal infections : impetigo ( 농가진 ), folliculitis( 모낭염 ), furuncles( 뽀로지 ), and carbuncles( 옹종 , 큰종기 ). .

Folliculitis : pyogenic infection in the hair follicles follicle is raised and reddened- small collection of pus beneath the epidermal surface. - Furuncles (boils) : extension of folliculitis, are large, painful, raised nodules

Carbuncles : furuncles coalesce and extend to the deeper subcutaneous tissue - chills and fevers, indicating the systemic spread of staphylococci via bacteremia to other tissues.

Impetigo : superficial infection that mostly affects young children, -a small macule (flattened red spot) - a pus-filled vesicle (pustule) on an erythematous base develops. - Crusting occurs after the pustule ruptures.

Figure 21-7 Staphylococcus aureus carbuncle. This carbuncle developed on the buttock over a 7- to 10-day period and required surgical drainage plus antibiotic therapy. (From Cohen J, Powderly WG: Infectious diseases, ed 2, St Louis, 2004, Mosby.)

Staphylococcal wound infections : in patients after a surgical procedure or after trauma- edema, erythema, pain, and an accumulation of purulent material. - the foreign matter removed, and the purulence drained.

Bacteremia ( 균혈증 ) and Endocarditis ( 심내막염 )

S. Aureus - bacteremia- infection of the lungs, urinary tract, or gastrointestinal tract -use of a contaminated intravascular catheter.

Acute endocarditis - S. aureus : a serious disease, mortality rate ~50%. -nonspecific influenza-like symptoms, disruption of cardiac output - 외과적 수술 후 또는 정맥내 삽관후 발생하는 원내 감염

Pneumonia ( 폐렴 ) and Empyema ( 농흉 )Aspiration pneumonia ( 흡식폐렴 ) : young, the elderly, and patients with cystic fibrosis, influenza, chronic obstructive pulmonary disease, and bronchiectasis

Hematogenous pneumonia is common for patients with bacteremia or endocarditis

necrotizing pneumonia with massive hemoptysis, septic shock, and a high mortality rate has been observed in recent years.

Empyema occurs in 10% of patients with pneumonia, - drainage of the purulent material is sometimes difficult.

Osteomyelitis ( 골수염 ) : Destruction of bones, particularly the metaphyseal area of long bones Septic arthritis ( 화농성 , 세균성 관절염 ): Painful erythematous joint with collection of purulent material in the joint space

Staphylococcus epidermidis and Staphylococcus epidermidis and other coagulase-negative Staphylococciother coagulase-negative Staphylococci

• Staphylococcus epidermidisStaphylococcus epidermidis

• Staphylococcus lugdunensisStaphylococcus lugdunensis

• Coagulase-negativeCoagulase-negative

• Catheter and shunt infectionsCatheter and shunt infections

• Prosthetic joint infectionsProsthetic joint infections

• Urinary tract infectionsUrinary tract infections

• diarrheadiarrhea

Identification (Identification (Staphylococcus aureusStaphylococcus aureus))

• hemolytichemolytic – sheep blood agarsheep blood agar– yellow pigmentedyellow pigmented (aureus)(aureus)

• mannitol fermentationfermentation• coagulase-positivecoagulase-positive

Staphylococcus epidermidis

• major component, skin floramajor component, skin flora

• opportunistic infectionopportunistic infection - less common than - less common than S.aureusS.aureus

• nosomial infections nosomial infections - shunts, cathetersshunts, catheters

• artificial heart valves/joints artificial heart valves/joints

IdentificationIdentification

• Non-hemolytic Non-hemolytic – sheep blood agarsheep blood agar– Non-pigmentedNon-pigmented

• Does not ferment mannitolDoes not ferment mannitol• Coagulase negativeCoagulase negative

Staphylococcus saprophyticusStaphylococcus saprophyticus• urinary tract infectionsurinary tract infections

• coagulase-negative coagulase-negative – not usually differentiated from not usually differentiated from S. epidermidisS. epidermidis

Antibiotic therapyAntibiotic therapy

• Resistance to penicillinResistance to penicillin– penicillinasepenicillinase

- lactam antibiotics (including methicillin) - lactam antibiotics (including methicillin) – often ineffectiveoften ineffective– modified penicillin binding proteinsmodified penicillin binding proteins

• Vancomycin Vancomycin • current drug of choicecurrent drug of choice• resistance has been observedresistance has been observed

Antibodies (monoclonal) Antibodies (monoclonal) - - MSCRAMM (microbial surface components recognizing adhesive matrix molecules) proteins : clumping factor

GRAM POSITIVE COCCI

S. aureus hemolyticmannitol yellow

+ -Staphylococcus (Clusters) Streptococcus (pairs & chains)

Catalase

BETA: Bacitracin S.pyogenes (group A)

CAMP/ Hippurate S. agalactiae (group B)

HemolysisCoagulase

S. epidermidisnonhemolytic (usually)mannitol white

ALPHA: Optochin /Bile Solubility S. pneumoniae

GAMMA: Bile Esculin 6.5% NaCl Group D* Enterococcus

Bile Esculin 6.5% NaCl Group D* Non-Enterococcus

(*can also be alpha hemolytic)

Note: S. viridans is Is alpha hemolytic and negative for all the tests below below

+

++

++

+

+

-

-

Summary Figure (Identification Scheme)

Enterococcus Group D streptococcus

• Growth on bile esculin agar– black precipitate

• 6.5% saline, 40% bile salts• grow

– enterococci• no growth

– non-enterococci

Groupable streptococci streptococci

• A, B and D– most important

• C, G, F – rare

Enterococci• distantly related to other streptococci• genus Enterococcus • catalase-negative, gram-positive cocci • gut flora

– urinary tract infection • fecal contamination

– opportunistic infections• particularly endocarditis

• most common E. faecalis

BOX 21-1. Important EnterococciOrganism Historical Derivation

Enterococcus enteron, intestine; coccus, berry (intestinal coccus)

E. faecalis faecalis, relating to feces

E. faecium faecium, of feces

E. gallinarum gallinarum, of hens (original source was intestines of domestic fowl)

E. casseliflavus casseli, Kassel's; flavus, yellow (Kassel's yellow)

Virulence Factor Biologic EffectSurface AdhesinsAggregation substance Hairlike protein embedded in cytoplasmic membrane that facilitates plasmid

exchange and binding to epithelial cellsEnterococcal surface protein Collagen-binding adhesin present in E. faecalisCarbohydrate adhesins Present in individual bacterium in multiple types; mediate binding to host cells

Secreted FactorsCytolysin Protein bacteriocin that inhibits growth of gram-positive bacteria (facilitates

colonization); induces local tissue damagePheromone Chemoattractant for neutrophils that may regulate inflammatory reaction

Gelatinase Hydrolyzes gelatin, collagen, hemoglobin, and other small peptides

Antibiotic ResistanceMultiple plasmid and chromosome genes

Resistant to aminoglycosides, β-lactams, and vancomycin

Table 23-3. Enterococcal Virulence Factors

BOX 243-2. Summary of Enterococci•Physiology and Structure Gram-positive cocci arranged in pairs and short chains (similar to Streptococcus pneumoniae) Facultative anaerobe Cell wall with group-specific antigen (group D glycerol teichoic acid)

•Virulence Factors Refer to Table 23-3

•Epidemiology •Colonizes the gastrointestinal tracts of humans and animals •Cell wall structure typical of gram-positive bacteria, which makes it able to survive on environmental surfaces for prolonged periods •Most infections from patient's bacterial flora; some caused by patient-to-patient spread •Patients at increased risk include those hospitalized for prolonged periods and treated with broad-spectrum antibiotics (particularly cephalosporins, to which enterococci are naturally resistant)

•Diseases• Urinary tract infections •Wound infections (particularly intraabdominal and usually polymicrobic) •Bacteremia and endocarditis

•Diagnosis •Grows readily on common, nonselective media. Differentiated from related organisms by simple tests (catalase negative, PYR positive, resistant to bile and optochin)

•Treatment, Prevention, and Control •Therapy for serious infections requires combination of aminoglycosides with a cell wall-active antibiotic (penicillin, ampicillin, or vancomycin); newer agents include linezolid, quinupristin/dalfopristin, and selected fluoroquinolones •Antibiotic resistance is becoming increasingly common, and infections with many isolates (particularly E. faecium) are not treatable with any antibiotics •Prevention and control of infections require careful restriction of antibiotic use and implementation of appropriate infection-control practices