Staphylococci

„The Grapes of Wrath“

Jan Tkadlec

Department of Medical Microbiology

18. 3. 2020

Staphylococci

• more than 50 species

• Common inhabitants of the skin and mucous membranes of animals (mainly mammals)

• Staphylococcus aureus – most important species

• Coagulase negative Staphylococci (CoNS) – majority of species but clinically less important

• S. epidermidis, S. saprophyticus, S. haemolyticus, S. capitis etc.

• Mostly part of normal microbiome, opportunistic infections, biofilm, foreign body infections

Know your enemy -

Biological properties of Staphylococci

„If you know the enemy and know yourself, you need not fear the result of a hundred battles.“

Sun Tzu: Art of War

Microscopic appearance

• Cell morphology - coccus

• 0,5-1,5 µm in diameter

• Gram-positive (purple)

• Arranged in grapes-like clusters (staphylé = bunch of grapes)

• May have a capsule

Electron microscopyLight microscopy - Gram staining

Clusters are formed as a result of cell division mechanism

Cultivation of Staphylococci• Easy to grow on common media

• Tolerate high salt concentration (up to 10%)

– Selective culture – manitol salt agar

– NaCl prevents growth of non-staphylococcal species, while fermentation ofmanitol (color change) differentiate S. aureus (yellow) from CoNS

Staph on blood agar

Biochemical properties• facultative anaerobes

– fermentation is primary source of energy generation

• grows in presence of oxygen– detoxification of reactive oxygen species

Staphylococci are catalase positive• Differentiation from streptococci (catalase negative)

Coagulase

• Enzyme that allows conversion of fibrinogen (soluble) to fibrin (insoluble)

• Two forms– Free – secreted in the surroundings

– Bound coagulase – attached to the surface of the bacteria cell

• Coagulase forms fibrin envelope around site of infection – protection from immune system

• Important for abscess formation

Production of coagulase – key to staphylococci differentiation

Fibrin capsule hides bacterial antigens.Prevents migration of immune cells.Allows to reach high concentration of bacterial toxins (cytolytic), that destroy immune cells (PMNs).

• Tube coagulase test:free coagulase

– Rabbit plasma inoculated with staphylococcalcolony, incubation 37°C for 1.5 hours

• Slide coagulase test (Clumping factor) –detection of bound coagulase

• Coagulase detection by latex agglutination

Detection of coagulase

Coagulation

Visible clusters

S. aureus

Biochemical differentiation of Staphylococci

Production of coagulase differentiate S. aureus (+) from other staphylococci that are clinically less important (mostly)CoNS – coagulase negative staphylococci

Note: there are rare strains of S. aureus that did not produce coagulaseOther coagulase positive species includes: S. delphini, S. hyicus, S. intermedius, S. lutrae, S. pseudintermedius, and S. schleiferi subsp. Coagulans, however these are rather rare in human clinical material

Important features of Staphylococci

S. aureus

• Loaded with toxins and other virulence factors– Adapted to actively cope with IS

• Broad spectrum of infections

• Severe infections (incl. sepsis, endocarditis)

• Resistance to ATB

CoNS

• Few virulence factors

• Even more resistant – ATB

• Form biofilm on foreignbody surfaces

• Low virulence but hard to eradicate

Staphylococci are everywhereOpportunity makes the thief

Sources - humans, animals, contaminated surfacesResistance – hard to eradicate the source• Antibiotics (ATB) and disinfectants• Environment – heat, desiccation, high salt concentration

• Survive several weeks (months) on contaminated surface

Staphylococcus aureus

• Coagulase positive!

• Major human pathogen

• Grows in large, round opaque colonies

• Golden pigment - aureus=golden– Presumed virulence factor (CoNS are non pigmented)

• Obligate / Opportunistic pathogen– Often infect immunocompromised patients but also healthy adults

– Loaded with the toxins and virulence factors

• Resistance to antibiotics (MRSA)

• No vaccination

• Health care associated infections

Recent taxonomy changes

• Some strains of S. aureus were recentlyreclasified as new species:

• Staphylococcus argenteus – clinicaly similar to S. aureus, also methicilin resistance

• Staphylococcus schweitzeri – rarely found in human

• Should be refered to clinicians as S. aureus complex, or at least explained that these species are not less severe than S. aureus, and are not CoNS

S. aureus carriageCarriage rate varies 10 - 60% of healthy adults

1. Persistent carriers – higher amount ofbacteria for longer time

2. Intermitent carriers – lower amount ofbacteria for shorter time

3. (Non-carriers)

– Varies with geographic location, ethnicity, age, sex and body niches

– Risk factors for Staph carriage:• Hormonal contraceptives, crowding and

healthcare exposure, low vitamin D status, atopic dermatitis, male gender, diabetes

– Protective role of S. aureus carriage againstseverity of future Staph infection -modulation of immune response

– S. aureus carriage - bacterial competition• S. epidermidis, Finegoldia magna,

corynebacteria• S. pneumoniae

Body niches colonised in S. aureus carriers

• Colonisation is risk factor for the S. aureus infection– Surgery, hospitalization, wounds

• Decolonisation:– Especially for MRSA carriers

Colonization staph is present in or on the body but is not causing illness.

Infectionstaph is present and causing illness.

Assault weapons

Virulence factors of S. aureus

S. aureus virulence factors• Broad spectrum of virulence factors• Mostly carried by the Mobile Genetic Elements• Variable, each strain has unique combination of

virulence factors

• Categories– Structural components

• Protein A, clumping factor, teichoic acid, etc.

– Enzymes• Staphylokinase (Fibrinolysin), Dnase, coagulase, catalase etc.

– Toxins• Cytotoxins, superantigens, exfoliatins

Virulence factors

• Function (categories):

– Adherence to host tissue – Adhesins

– Tissue specific damage, and spread – some toxins

– Interference with host immune function – evasins, also toxins

Interference with phagocytosis

S. aureus could survive and escape from phagocytic cells

S. aureus as an intracellular pathogen• Protection from immune system and antibiotic treatment

• Survival of S. aureus within:

– non-phagocytic cells - endothelial cells, epithelial cells, keratinocytes, fibroblasts and osteoclasts• Facilitate persistence and recalcitrance of infection

– Macrophages and neutrophiles – „trojan horse“ for spread of staph through thebody• Lower rate of S. aureus bacteremia in neutropenic patient – lack of neutrophiles

superantigens (sAgs) enterotoxins and toxic shocksyndrome toxin-1 that bind the MHC class II receptor to T- cell receptors – leading to strongactivation of the T cells

protein A, binds the Fc region of antibody and the Fab regions of the B-cell receptor - block opsonophagocytosis and cause B-cell death

protein Map - the MHC class II analogue, whichbinds the T-cell receptor (TCR) and modulatesimune response

Evasion of the immunoresponce by S. aureus

S. aureus is highly adapted to cope with humanimmune system

Toxins

• Intoxication is not necessary infection– Toxins are cause of symptoms not living bacteria

1. Cytolytic toxinsa) Hemolysins

b) Leucocidins (PVL)

2. Enterotoxins

3. Toxic shock syndrome toxin (TSST)

4. Exfoliatins

Superantigens

Cytotoxins:

• Haemolysin α (Hla): pore-forming, cytotoxic to many types of cells including muscle cells, but not neutrophils

– Complete haemolysis (looks like beta haemolysisin streptococci)

– Main virulence factor – SSTI, sepsis, pneumonia

• Haemolysin β: degrades sphingomyelin and is toxic for many kinds of cells, including human RBCs.

PVL toxin

• Panton-Valentin Leukocidin (1932)

• Bicomponent pore-forming toxin

– the LukS-PV and the LukF-PV subunits

– Targets: neutrophils, monocytes and macrophages

• High host specificity

– Induce rapid activation and cell deathin human (and rabbit) neutrophils

– But didnt affect murine or simian cells

– 2-3% of S. aureus clinical isolates

PVL in pathogenesis

Acute, severely necrotising skin infections and necrotising pneumonia

• Pneumonia often preceded by influenza

• High mortality, rapid progress

PVL gene mostly in community acquired S. aureus and MRSA (CA-MRSA) infections

PVL action in necrotising pneumoniaInflammation (influenza) cause migration and accumulation of PMNs in lungs.Production of PVL by S. aureus cause rapid lysis of PMNS and release of toxic antimicrobial compounds.Released serine proteases starts to digest lung tissue causing massive damage

Superantigens (SAgs)

• Non-specific massive activation of the T-cells

Release of cytokines

– Can lead to septic shock

– Enterotoxins

– TSST

Food poisoning• Enterotoxins are chemoresistant and heat-stable

secreted polypeptids– More than 20 genes – sea, seb, sec…

• Clinical isolate has on average 6 enterotoxin genes

– It can withstand boiling at 100°C for a few minutes– Infected food (milk products, meat, ice cream, …)

• Incubation period 2-6 hours• Interfere with intestine function• Clinical symptoms - nausea, vomiting and

diarrhea• Self limited, recovery in day or so

Toxic Shock Syndrom

• TSST 1 (Toxic Shock Syndrome Toxin)

• Toxin producing S. aureus is usually localized atmucosal sites (nasopharynx, vagina – tampons) orin abscesses

• released toxin circulate in blood and stimulaterelease of IL-1, IL-2, TNF-α and other cytokines

• high fever, rash, desquamation, diarrhea, vomiting, hypotension even multiple organ failure

• Fatal outcome isn´t rare

Scalded skin syndrome• Exfoliatin - epidermolytic toxin

• ETA, ETB, ETD

– Serine proteases – „molecular scissors“ specifically cut the human desmoglein 1 –keratinocyte cell-cell adhesion molecule

– Leading to the disruption of the top layers of the skin and facilitating bacterial skin invasion

Infections caused by Staphylococci

Where you could expect Staphylococci

UrineUTI: S. aureus (catheter related)S. saprophyticus

Nose or throat (Swabs) CoNS – colonizationS. aureus colonization or upper airways infection

Heart (valve tissue)Infective endocarditis:S. aureus or CoNS

StoolS. aureus rarely cause colitis

Blood (bloodculture)BSI: S. aureusCoNS (catheter related)

Skin (swabs, pus, tissue)S. aureus SSTI orcolonisationCoNS - colonisation

Osteoarticular infections (pus, tissue)Osteomyelitis, septic arthritis, prostheticjoint infectionS. aureus and CoNS (implant related)

Lungs (sputum)PneumoniaS. aureus

S. aureus toxicosesEnterotoxicosis – food poisoningToxic shock syndrom

CSFMeningitidisS. aureus and CoNS

Source of staphylococal infection:

– Endogenous – colonization precedes infection (majority of the cases)

• Decolonization before surgery!

• Localized and systemic infections

• From benign to live-threatening

Pathogenesis of S. aureus infections

Colonisation Localised SSTI

Invasion of blood stream

Bacteremia

Sepsis

Organ infection or abscess Osteomyelitis• Endocarditis• Pneumonia• Other organs

wound

Another localisedSSTI

S. aureus infections are often metastatic- chronicity and recurence

Stages of S. aureus infection

Disemination could lead to bloodstream infection and infectionof other organ systems

Attachment Localised infection Disemination

Why are Staphylococci clinically so important?

Staphylococcus aureus is leading causse of Skin and Soft Tissue Infections (SSTI)

MSSA –methicillin sensitive S. aureus; MRSA –methicillin resistant S. aureus;

Skin and soft tissue infections

• S. aureus is most common cause of SSTI

– Cutaneous abscess (folliculitis, carbuncles, furuncles) is hallmark infection of S. aureus SSTI

Folliculitis

Folliculitis

Furuncle (boil)

Carbuncle

Impetigo Paronychia

CellulitisWound infection

Impetigo

Impetigo is the most common bacterial skin infection of children. In general, impetigo presents as bullous or papular lesions that progress to crusted lesions, withoutaccompanying systemic symptoms, on exposed areas of the body (usually the face or extremities).

Other SSTI• Necrotizing fasciitis – severe rapidly

progresing infection of the fascialplane deep to the subcutaneous tissue

• Pyomyositis – purulent infection of skeletal muscle that arises from hematogenous spread, usually with abscess formation, tropical countries

• Wound infection and surgical siteinfections– postoperative mediastinitis –

complication of cardiac surgery– Nosocomial infections

• Mastitis - infection of one or both of the mammary glands

Increasing trend in numbers and severity of staphylococcal SSTI

• USA but also other countries

• Associated with rise of the community associated MRSA since late 1990s

• CA-MRSA carries often PVL – more severe infections

• number of emergency department (ED) visitsfor SSTIs in the United States increased from 1.2 million in 1993 to 3.4 million in 2005

Treatment and diagnosis of SSTI

• drainage if there is drainable focus• In case of uncomplicated SSTI antibiotics are maybe

not necessary• complicated cases: oral or parentheral antibiotic

therapy – co-trimoxazole, antistaphylococcalpenicilins, clindamycin. In case of MRSA: vancomycin, daptomycin, linezolid.

• Superficial infection (impetigo) could be treated by topical treatment (fusidic acid or mupirocin)

• Dx: swabs or collected pus

Bloodstream infections (BSI)

S. aureus bacteremia (SAB)• Incidence 10-30 per 100,000 person-years• Risk factors:

– Age –highest risk up to first year of life and above 70 years– Gender – males higher chance of SAB– Ethnicity – black population in US and indigenous Australians has

increase risk of SAB– HIV – as much as 24 times increase risk of SAB compare to non-HIV

population– Injection drug users – non-sterile needles– S. aureus colonisation – prevention via decolonization before surgery,

etc.– Hemodyalisis patients (catheters)

Primary foci of SAB– catheter, SSTI, pulmonary inf., osteoarticular inf., endocarditis

• Mortality depending on foci ranges from 10 (SSTI) to 70 % (pulmonary)

• Treatment : i.v. betalactams and glycopeptides (vancomycin), daptomycin, etc.

Infective endocarditis

Infective endocarditis (IE)

• Staphylococci form a biofilm on the hearthvalve (native or prosthetic), bacteria are release into the blood and could cause infection of other organs

• S. aureus is most common cause of IE

• Risk factor is healthcare contact, injection drug use, SAB, stroke, diabetes

• Treatment: prolonged i.v. therapy

Osteoarticular infections

Osteomyelitis

Osteoarticular infections

Infection via haematogenous spead or a direct trauma

S. aureus is most common cause

Osteomyelitis

Septic arthritis

Prosthetic joint infection

Often chronic and recurent, requires prolongedantibiotic treatment and often surgery

Pneumonia

• Community acquired pneumonia (CAP)– superinfection after influenza, severe when PVL toxin

production – necrotizing pneumonia

• Hospital acquired pneumonia (HAP)• Ventilator associated pneumonia (VAP)

S. aureus is common cause in all three types

• CAP and HAP – secondary pneumonia=hematogenic spread from other foci of infection

Foreign body infection

• Cardiac device infections

• Intravascular catheter infections

• Breast implants

• Ventricular shunts

• Other foreing bodies

Clinical impact of coagulase-negative Staphylococci

• Frequent on the skin and mucous membranes

– Often a contamination of the clinical samples

• Opportunistic pathogens -immunocompromised patients

• Relatively low virulence

• Resistance and often biofilm formation

CoNS could cause broad range of infections similar to S. aureus, however less severe:

Bloodstream infection (catheter related), endocarditis, wound infections, osteoarticular infections, etc.

S. epidermidis, S. haemolyticus and S. hominis are most frequently found in human clinical samples

S. saprophyticus – urinary tract infections in youngwomen

Hard to killAntibiotic resistance in S. aureus

How to became a Superbug: story ofthe resitance of S. aureus

„If we are not careful, we will soon be in a post-antibiotic era…and for somepatients and for some microbes, we are already there.“ - Tom Frieden; CDC

„Superbugs Could Kill 10 Million Each Year By 2050.“ – Jim O´Neil; Review on Antimicrobial Resistance .

ESKAPE pathogens

• Enterococcus faecium, Staphylococcus aureus, Klebsiella

pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and

Enterobacter spp

„….they currently cause the majority of hospital infections and effectively “escape” the effects of antibacterial drugs.“

First step: resistance to penicillin

• Penicillin (Fleming 1928, introduction 1940s)

• S. aureus infection mortality was about 80% before introduction of penicillin

• Penicillin resistence in mid 1940s

• Enzyme penicillinase – plasmid encoded– Hydrolysis of the beta - lactam ring

– Resistance to penicillin G, ampicillin and similar drugs

• Now penicillinase producing strains highlyprevalent

Second step: MRSA

• Introduction of methicillin (1956)– Semisynthetic derivate of penicillin

– Resistant to penicillinase

• MRSA (methicillin resistant S. aureus) 1961– Resistance to penicillin, methicillin (oxacillin) and

cefalosporins

– often resistant to tetracyclines, macrolides and aminoglycosides

– Susceptible to vancomycin, linezolid, daptomycin

– Higher mortality and morbidity compare to MSSA

Mechanism of MRSA resistance

In susceptible bacteria beta-lactams binds PBP2 (penicillin binding protein) and prevents formation of peptidoglycan crosslinks, leading to disruption of cell wallIn MRSA beta-lactams are unable to bind PBP2a (coded by mecA gene)

Prevalence of MRSA around the world

Europe MRSA among invasive isolates

Decrease of MRSA in Europe

UK: Active measures to decrease MRSA

BUT the numbers represents prevalence (i.e. proportion of MRSA among S. aureus infections)and incidence is growing (total numbers of MRSA cases)

Epidemiology of MRSA

• Clonal complexes (CC)– geneticaly separated lineages within S. aureus population

– Diference in MGE content – specific virulence and resistence features

– About ten major lineages e.g CC5, CC8, CC30…

• Typing methods to identify lineages– MLST (MultiLocus Sequence Typing – STs sequence types)

– SCCmec typing

– spa typing

• Clone assignment based on MLST an SCCmec type– E.g ST5-MRSA-I

• MRSA according to their origin:– Healthcare-associated (HA-MRSA)

• Multiple resistance

• nosocomial or hospital infection

• Major form of MRSA until 1996

• Nosocomial pneumonia

• Catheter related UTI

• BSI

• SSTI

• E.g. CC5, CC30

– Community-associated (CA-MRSA)• Often only methicilin resistance

• Severe SSTI and necrotizing pneumonia

• Often PVL toxin

• Growing prevalence

• Children, adults and otherwise healthy people

• Prevalent in USA and Asia

• CC8 (USA300), CC80, CC59

Old McDonald had a farm…

Livestock-associated MRSA

• Animals as a source of infection– Pigs, cattle, poultry– Asymptomatic cariage or SSTI

• Persons in direct contact with animals– farmers, slaughterhouse workers etc.– Could cause serious infections like pneumonia, sepsis or severe SSTI

• Specific animal lineages• PVL in some lineages• Tetracycline resistence is typical – use in agriculture• CC398 (from pigs), CC9 …

Interestingly, dogs and cats are colonized/infected by human lineages

Third step: VISA, VRSA/GRSA

• Resistance to glycopeptides (vancomycin)

• VISA – Vancomycin intermediate, Japan 1996

• VRSA/GRSA – Vancomycin/glycopeptideresistant S. aureus

• Transfer of the vanA gene from enterococci

• Fortunately, until now only sporadic cases.

VRSA resistance mechanism

• Vancomycin binds to the two D-ala residues on the end of the peptide chains and preventcross-links formation

• In resistant strains D-ala are replaced by D-lactate, so vancomycin cannot bind.

VISA resistance

• Thicker cell wall – more binding sites forvancomycin, higher concentration is needed.

Evolution of S. aureus resistance

Unfortunate story of S. aureus vaccinedevelopment

• Vaccination is clever solution for antibiotic resistance, and prophylaxis

• Why there is no vaccine against S. aureus?• Lack of effort? – No, dozens of vaccines tested in last

twenty years• So far, any candidate vaccine did not demonstrate

efficacy.• Reasons:

1. One target is not enough (unlike capsule in pneumococci)

2. Variation in target molecules (different strains)3. Different target for different disease (e.g.. SSTI vs sepsis)4. Host genetics5. Immunopatological reactions may occur

Vaccines that reached clinical testing

• StaphVax – testing in humans does not show protection from S. aureus infection

• V710 – failed to elicit the immunity– Even worse, vaccinated patients that get infected

have worse outcome

– all 12 V710 recipients (but only 1 of 13 placebo recipients) with low IL2 levels prior to vaccination and surgery died after postoperative S. aureus infection

Additional sources

Review S. aureus infectionshttps://cmr.asm.org/content/28/3/603.longReview MRSAhttps://www.nature.com/articles/nrdp201833Guidelines for MRSA treatmenthttps://academic.oup.com/cid/article/52/3/e18/306145

Thank you for your attention!