Staphylococci

• Staphylococcus (in Greek staphyle means bunch of

grapes and coccos means granule) is a genus of Gram-
positive bacteria

• Under the microscope they

appear round (cocci), and
form in grape-like clusters.

• The Staphylococcus genus

includes thirty-one species.

• Most are harmless and

• reside normally on the skin and mucous membranes of
humans and other organisms.

• Found worldwide, they are a small component of soil

microbial flora.
• Really only 3 main species that infect humans

• Most pathogenic being S. aureus which is

becoming increasingly problematic among
healthy individuals

• S. epidermidis is a problem mainly associated

with immunocompromised patitnet especially
those with indwelling catheters

• S. saphrophyticus which causes UTIs in sexually

active females (aka honeymooners cystitis)
• S. aureus is well documented as a human
opportunistic pathogen.

• It is present as part of the normal flora in the nose of

30% of the human population

• Some of the more serious infections of S. aureus are

bacteraemia, pneumonia, osteomyelitis, meningitis,
acute endocarditis and toxic shock syndrome.

• Food poisoning is often attributed to staphylococcal

enterotoxin.
• The sites and severity of Staphylococcal infections vary
hugely.
• Largely considered to be opportunistic: S. aureus is a major
cause of hospital acquired (nosocomial) infection of surgical
wounds and infections associated with indwelling medical
devices.

Lowy, F.
Staphylococcus aureus infections.
N Engl J Med. 1998 339:520-32.
As an opportunistic pathogen how does it go
from colonising your nose to killing you?
Minor skin infections Impetigo: common
where the bacteria in young children
gain access to hair
follicles

More serious scalded skin syndrome.

Symptoms are a result of a specific toxin:
Exfolative toxin A (Eta)

Sinusitis also when bacteria enter

Sinuses causing inflammation.

Stye on eyelids where eyelash follicles

become infected
• Food poisoning: action of specific toxins,
not usually live bacteria (enterotoxins)

• Toxic shock syndrome: (Tsst-1 toxin).

Tampon associated shock: superantigen

• Pneumonia: often occur after influenza

infections, high mortality rates.
 Systemic disease
• Presence of bacteria in blood is often enough to kill as
the toxic shock (inflammatory response) can overwhelm
body.

• But they can then move from blood and infect other
organs (role of FnBPs and Eap discussed later)

• Secondary abscesses in brain, kidney etc

• Endocarditis, when heart tissue directly infected

• Osteomyelitis: chronic infection of bone.

 Attachment/adherence
• Many proteins expressed on surface of S. aureus that
allows it to attach to human tissues:

• Two major proteins Fibronectin-binding protein and the

fibrinogen-binding protein (aka clumping factor).

• Two versions found in many S. aureus strains (A and B)

• Que, Y.A. et al. Fibrinogen and fibronectin binding

cooperate for valve infection and invasion in
Staphylococcus aureus experimental endocarditis. J Exp
Med. 2005 May 16;201(10):1627-35.

• Also both involved in platelet activation which leads to

blood clotting
 Invasion
• For many years S. aureus was considered and
extracellular pathogen.

• Now know to have the ability to invade cells

• Two major clinical features of this are the development

of endocarditis

• And the exit of bacteria from the blood stream where the
bacteria attached to and invade endothelial cells
( ref: How does Staphylococcus aureus escape the bloodstream? Edwards AM,
Massey RC. Trends Microbiol. 2011 Apr;19(4):184-90.)

• The Fibronectin-binding protein is responsible for this

 Fibronectin binding protein:
S. aureus’ invasin
s A 1 2 3 4 5 6 7 8 9 10 11 W M

Staphylococcus aureus

FnBPs
Fn

integrin

Host cell membrane

Basic model of the protein:protein interactions

that lead to invasion of host cells by S. aureus.
Fn: fibronectin; FnBPs: fibronectin binding proteins.
 Toxins
• Major factor in S. aureus pathogenicity
• Endless list that varies from strain to strains as many of
them are found on mobile genetic elements such as
pathogenicity islands and phages.

• TSST-1
• Enterotoxins (A-J)
• Exfolative toxins (A and B)
• Alpha, beta, delta and gamma toxins
• Panton-valentine leukocidin
 Tsst-1: superantigen

Enterotoxins are also superantigenic but they act in gut resulting in massive inflammation
Exfolative toxin is a
protease that cleaves
desmoglein (Dsg) in
epidermis resulting in
disruption of skin
structure and Scalded
skin syndrome
Alpha, beta, gamma and delta

Alpha and delta are Beta is a sphingomyelinase

pore forming toxins
Gamma is a leukocidin:
specifically lyses leukocytes

Cell membrane
 Pvl toxin
• Panton Valentine leukocidin toxin is a bi-component, pore-forming
exotoxin that targets cells of the immune system such as
neutrophils.

• The active form of PVL requires the assembly of two polypeptides,

LukS-PVand LukF-PV, into a heterooligomeric pore.

• The genes encoding the components are found on a phage (can be

horizontally acquired)

• This gene is commonly found in association with CA-MRSAs and

believed to be a contributor to the increased virulence of these
strains of MRSA.
Regulation of expression of virulence factors
The accessory gene regulatory locus (Agr)
 At low cell densities the At high cell densities the
concentration of the peptide concentration of the peptide
is low so the cells express is high so the cells express
their adhesins their toxins

This is believed to allow the bacteria to “stick” during the initial phases of infection
And once they have established themselves express their toxins which will allow
them to release more nutrients and move onto a new site
 Innate and adaptive immunity
(1) Anatomical: skin and mucous membranes

(2) Physiological: temperature, pH, lysozyme,

interferon, complement

(3) Phagocytic barriers:

(4) Inflammation

(5) B and T cells

 Skin and mucosal membranes?
• With 30% of human population as carriers skin must be
in the vast majority of cases successful at protecting us.

• But the bacteria have evolved to specifically bind to

keratinocytes (fibrinogen binding protein)

• If they enter hair follicles they can form abscesses

• They seem to (in vast majority of cases) depend upon a

breach in this to cause infections
 Physiological: temperature, pH,
lysozyme, interferon, complement
• Temperature: almost always found in association with a
warm blooded animal, optimum growth temp is 37oC

• pH…gut survival isn’t a major feature of disease

• Lysozyme: cleaves peptidoglycan, and can be protective

for host, but thick peptidoglycan cell wall makes them
more resistant than Gram –ves

• Interferon: not yet known if this is protective

• Complement: SCIN
• Inactivation of complement.

• Assembly of C3 convertases on the surface of bacteria is

a prerequisite for complement activation.

• Specific complement proteins carry out the essential

function of cleaving C3, which results in release of
soluble C3a and covalent attachment of C3b to the
bacterium.

• S. aureus secretes a 9.8-kDa protein called

Staphylococcus complement inhibitor (SCIN), which
binds to and stabilizes these proteins, resulting in
inhibition of further C3b formation
• Resistance to phagocytosis
• The ability of S. aureus to avoid opsonins present in normal
serum is an important factor in the success of infection.

• Antibodies that recognize cell-surface components such as

teichoic acid, peptidoglycan and surface-associated
proteins are present in sera of most, if not all, individuals.

• S. aureus expresses surface-associated anti-opsonic

proteins and a polysaccharide capsule that can both
interfere with the deposition of antibodies and complement
formation by classical and alternative pathways, or with their
access to neutrophil complement receptor and Fc receptor.

• Therefore, efficient phagocytosis by neutrophils that

requires recognition of bound complement and antibody is
compromised.
• Capsule.
• Most S. aureus clinical isolates express a thin
microcapsular layer that is composed of serotype 5,
serotype 8 or serotype 336 capsular polysaccharide
•
• Expression of capsule is associated with increased
virulence in animal infection models

• In vitro phagocytosis assays revealed that the presence

of the capsule reduced the uptake of cells by neutrophils
in the presence of normal serum opsonins, indicating
that capsule is anti-opsonic

• Complement factors can assemble on the cell-wall

surface underneath the polysaccharide, but presumably
these are inaccessible to complement receptor on the
surface of neutrophils.
• Staphylococcus can coat itself in host proteins
disguising it from host

• Antibodies
• Fibronectin
• Fibrinogen
• Elastin
• Collagen
• Von Willebrand factor
• Keratin
• Etc…….
• Inhibition of neutrophil chemotaxis

• Immediately as the bacterium gains a foothold in the host and starts

to grow, several chemoattractants for neutrophil released

• The peptide fragments C3a and C5a, released by complement

activation, as well as formylated peptides secreted from growing
bacterial cells, are recognized at high affinity by specific
transmembrane G-protein-coupled receptors on the neutrophil
surface

• These are stimulated and activate intracellular signalling cascades,

resulting in migration of neutrophils from the blood to the site of
inflammation.

• About 60% of S. aureus strains secrete the chemotaxis inhibitory

protein of staphylococci CHIPS that can bind avidly to both the
formyl peptide receptor (FPR) and the C5a receptor (C5aR) to block
the cognate agonist from binding
Is inflammation protective against S. aureus
infections?
• Like many pathogens the inflammatory response is often
responsible for some of the observed pathogenicity
• In vast majority of skin/soft-tissue infections it is sufficient
to clear infection, but in systemic infections or in deeper
tissues/organs it can be very damaging
• S. aureus abscesses contain considerable amounts of
pus (bacteria and neutrophils)
• As the bacteria can avoid phagocytosis it may not be
protective?
• May provide the bacteria with a transmission route?
 B and T cells?
• S. aureus secretes an array of toxins that allow them to
directly lyse these cell types

• Everyone has anti-staphylococcal antibodies in their

blood but still get infections??

• There doesn’t appear to be any “memory” to secondary

infections, which may suggest they are not overly
protective
Foster, T.J. Immune evasion by staphylococci. Nat Rev Microbiol.
2005 Dec; 3:948-58.
 Biofilm formation

• Biofilms consist of microorganisms immobilised on a surface embedded in

an organic polymer matrix (slime)
• Protects bacteria from washing away and slime protects from antibiotics
and immune system
• Slime can be made of up of varying quantities of carbohydrate, protein and
eDNA
 Treatment of staphylococci
• No vaccine available, but many in
development…many failures!!

• Antibiotics mainly used……but….

Penicillin   

Penicillin was the first

antibiotic that was
successfully used in
treating bacterial
infections.

Before its development,

many people suffered
and died from bacterial
infections that are no
longer considered
dangerous today.
 Resistance
Drug first
introduced reported

Penicillin
Streptomycin
Chloramphenicol
Tetracycline
Erythromycin
Vancomycin
Methicillin
Cephalosporins
Linezolid

1940 1950 1960 1970 1980 1990

95% of S. aureus population now resistant to Penicillin

MRSA rates (% bacteremia) in participating countries in 2012 (ECDC)
• Penicillin binding proteins (PBPs) are responsible for the final
stages of peptidoglycan synthesis (major component of cell
wall)
• Inhibition of
PBPs can result
in lysis of
bacterial cells
• Methicillin is a
derivative of
penicillin
(-lactam family
of antibiotics that
bind the PBPs to
lyse cells)
• MRSA strains have acquired a gene called mecA that encodes an
alternative PBP: PBP2a that can substitute activity in the presence of the
antibiotic
• The mecA gene is found on the SCCmec mobile genetic elements
• Hiramatsu K, Cui L, Kuroda M, Ito T. The emergence and evolution of
methicillin-resistant Staphylococcus aureus. Trends in Microbiology. 2001
Oct;9(10):486-93.
2001/2002 community acquired MRSA strains emerged.
• MRSA infections acquired in hospitals and healthcare settings
can be severe.

• Certain factors can put some patients at higher risk for

MRSA :
– prolonged hospital stay
– receiving broad-spectrum antibiotics
– being hospitalized in an intensive care or burn unit
– spending time close to other patients with MRSA
– having recent surgery
– or being a carrier of MRSA in the nose
 Cost of MRSA
• Directly attributal costs:
– “hotel costs”
– Cost of diagnostic procedures
– Morbidity and mortality after infection
– Glycopeptides (vancomycin) more expensive

• Cost of control
– Involvement of infection control team
– Temporary closure of wards and theatres
– Redeployment of staff

• In a UK outbreak of 30 patients over 5 weeks costs of £13,000

not including isolation costs or increased hospital stay
 Antimicrobial resistance is a major threat to
public health
• Research indicates that it takes scientists 3 years to develop a
new antibacterial drug, but it takes the bacteria only 3 months
to develop a resistance to that drug.

• Globally we need to look at how antibiotics are used and

reduce their inappropriate use

• Find new strategies to combat

infectious diseases
 Community-acquired MRSA (CA-
MRSA)
• Until recently (approx 10 years) MRSA infections have been confined to
hospital environments.

• One of the assumptions explaining this is that the fitness costs incurred by
being resistant meant that they were only competitive in environments with
immunocompromised patients

• It has recently been estimated that in the UK 2% of hospital admissions will

become infected with S. aureus, and of those 40-60% will be MRSA.

• In past 5 years new strains of MRSA have been emerging that Infect healthy
individuals with no prior hospital associations

• A highly virulent form known and community-acquired MRSA.

 HA-MRSA & CA-MRSA
HA-MRSA:
•Associated with prior exposure to a hospital/ healthcare setting.
•Contain larger type I, II & III SCCmec elements

CA-MRSA:
•Usually infects healthy individuals with no prior healthcare exposure-
no classical risk factors.
•Carry novel smaller type IV and V SCCmec elements.
•Appear to be more virulent than HA-MRSA strains.
•Lower MICs than HA-MRSAs- due to lowered mecA expression?
• PVL?

• Alpha toxin?

• Cytolytic toxins?

• Still much to learn about this pathogen

• Have these strains compensated for the fitness costs

associated with being resistant? Smaller mec element?
Collins et al. 2008. Identification of Factors Contributing to
T-Cell Toxicity of Staphylococcus aureus Clinical Isolates.
Journal of Clinical Microbiology, 46:2112-4

TABLE 1. Bacterial factors significantly associated with T-cell toxicity

Bacterial phenotype Mean (median) % T-cell survival No. of strains

Beta toxin positive 59.4 (61.9) 163
Beta toxin negative 74.5 (77.1) 42
Delta toxin positive 59.2 (61.7) 51
Delta toxin negative 72.4 (77.7) 154
TSST-1 positive 69.1 (72.4) 53
TSST-1 negative 60.4 (63.6) 151
MRSA 69.8 (77.2) 23
MSSA 61.6 (66.1) 183
Agr type 1 54.2 (56.6) 77
Agr type 2 66.3 (70.6) 47
Agr type 3 68.4 (72.1) 75
CC1, CC12, CC15, CC22, CC30, CC39, CC51 67.69 (67.9) 110b
CC5, CC8, CC9, CC16, CC25, CC45 55.57 (57.8) 93c

a For beta toxin positive versus beta toxin negative, P < 0.001; for delta toxin positive versus
delta toxin negative, P < 0.001; for TSST-1 positive versus TSST-1 negative, P = 0.009; for
MRSA versus MSSA, P = 0.045; for Agr type 1 versus Agr type 2, P = 0.005; for Agr type 1
versus Agr type 3, P < 0.001; for Agr type 2 versus Agr type 3, P > 0.1; and for CC1, CC12,
CC15, CC22, CC30, CC39, and CC51 versus CC5, CC8, CC9, CC16, CC25, and CC45, P < 0.001.
All P values were determined by the Mann-Whitney U test.

b CC1, n = 9; CC12, n = 6; CC15, n = 19; CC30, n = 57; CC39, n = 13; CC51, n =

Type II MRSA less toxic than
MSSA. p= 0.0003

Conclusion- carriage of type II SCCmec elements is associated with

reduced toxicity.
The mecA gene affects toxicity.

Conclusion- The mecA gene and more than likely the PBP2a peptide is
responsible for reduced toxicity in HA-MRSA.
Inactive Agr system in MRSA?
40000 BH1CC RNA III gfp

ΔSCCmec RNA III gfp

30000 ΔmecA RNA III gfp

RN6930B Agr+ RNA III gfp

20000
RN6119 ΔAgr RNA III gfp

10000

-10000
0 2 4 6 8 10 12
-20000

Conclusion- presence of the type II SCCmec element prevents

normal stationary phase activation of Agr.
How can mecA interfere with Agr?
Removal of cell wall restores Agr.

Conclusion- PBP2a changes the cell wall, blocking AIP recognition by

the Agr system.
Methicillin resistance reduces the virulence of healthcare-associated methicillin-resistant
Staphylococcus aureus by interfering with the agr quorum sensing system. Rudkin JK,
Edwards AM, Bowden MG, Brown EL, Pozzi C, Waters EM, Chan WC, Williams P, O'Gara
JP, Massey RC. J Infect Dis. 2012 Mar 1;205(5):798-806
 Why is this seen only in HA-MRSA and
not CA-MRSA?

Expression levels differ, HA-MRSA express significantly higher levels of mecA

 Can we affect this?

Rudkin et al. Antimicrob Agents Chemother. 2014

Feb;58(2):1100-7.
Specifically, which toxins are affected?

A: coomassie of total secreted proteins

B: Western using anti-PVL antibodies
C: Western using anti-alpha toxin antibodies
D: PSMs (phenol soluble modulins).
 Different effect on different cell types:

Therefore the effect is likely to be site specific

Koser et al 2012. N Engl J Med. 2012 June 14; 366(24): 2267–2275.
Rapid Whole-Genome Sequencing for Investigation of a Neonatal
MRSA Outbreak
They were able to show it was an outbreak by
plotting sequence data on a tree
Young et al 2012. Evolutionary dynamics of Staphylococcus aureus during
progression from carriage to disease. Proc Natl Acad Sci U S A. 2012 March 20;
109(12): 4550–4555.
 summary
• Malaria, TB, Salmonella, E. coli, S. aureus
• Disease/symptoms
• Virulence factors
• Immunity to infection
• Immune evasion
• Treatments and implications this may have on future
evolution of pathogen
• Specific genetic regulatory mechanisms
• Effect of mobile genetic elements
• Impact of whole genome sequencing