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!