CAMRSA Biological Characteristics

1
CA-MRSA Biological Characteristics

• SCCmec elements and PVL
• Susan Boyle-Vavra
• University of Chicago

2
Brief History of b-lactam resistance in S aureus

• 95 S. aureus isolates resistant to penicillin
  due to b-lactamase
• 1959 b-lactamase insensitive compounds
  Methicillin, nafcillin, oxacillin
• First methicillin-resistant isolate in 1960s
• Perceptible increase in MRSA not until 1980s
• MRSA comprises 60 of S. aureus infection in
  hospitals today

3
Increase in University of Chicago MRSA(Peds )
4
MRSA acquired low affinity penicillin-binding
protein, PBP2a (mecA gene)
Penicillin-binding assay

PBP2a
PBP2
PBPs
PBP2 and PBP2a act together
5
mecA gene carried on SCCmec
MSSA acquired the mecA gene encodes a PBP with
low affinity for penicillin (PBP2a) Carried on a
large chromosomal element (SCCmec) Integrates
into chromsome in orfX site specific int
20 kb-60kb
orfX
mecA (complex A, B and C)
ccr recombinase genes (allotypes A, B, C and
complex 1,2,3,4,5,)
6
Emergence of CA-MRSA in Chicago
Community-Acquired MRSA in Children With No
Predisposing Risk
Herold et al JAMA 1998 279593-598.
1988-1990 1993-1995

• 35
• 10 259

CA-MRSA disease (no risk) Prevalence (per
100,000 admissions)
a 156-bed tertiary care hospital, with
4800 admissions annually including a 22-bed
pediatric intensive care unit and a 52-bed
intensive care nursery.
7
Hospital Associated-MRSA

• Risk for infection includes contact w healthcare,
  previous antibiotic use, indwelling lines,
  surgery, long term illnesses etc
• are multiply resistant
• penR, OxR, GmR, CmR, eryR, clinda,

8
Community-Acquired MRSA in Children With No
Predisposing Risk
Herold et al JAMA 1998 279593-598.

• Lack traditional risk factors associated w MRSA
• Clinical syndromes the same as those of CA-MSSA
  (SSTIs, osteomyelitis etc)
• Tended to be pan-susceptible to non-b-lactams

9
SCCmec IV is associated with CA-MRSA

• Ito et al had just described SCC mec I, II, III.
• SCC mecII and III carried antibiotic resistance
  genes and were hospital associated
• We recognized that CA-MRSA were non-multiply
  resistant to non-b-lactams
• We hypothesized that the CA- MRSA SCCmec element
  might be different than the others
• Ma et al., sequenced SCC mec of our Chicago
  isolates

10
mec
ccr
SCCmec types
Adapted from Okuma et al., J Clin Microbiol. 2002
Nov40(11)4289-94.
pls
B
1
orfX
A
2
kdp
orfX
A
3
orfX
B
2
orfX
C2
ccrC1
Type V
orfX
11
SCCmec Type IV

• Smaller than SCCmec types I, II, III
• Lacked antibiotic resistance genes except mecA
• Circulating in multiple genotypes around the
  world
• (ST 1, 8, 30, 59, etc)
• Seemed to be entering into MSSA genetic
  backgrounds that were present in particular
  geographic locales
• Provided molecular evidence that CA-MRSA did not
  simply drift from the hospital into the community

12
New SCCmec type in CA-MRSAtype IV
Ito et al., Ma et al.,
SCCmec typing a consensus
Mec complex (ABCDE)
Ccr complex 1,2,3,4,5,
13
SCCmec integrates into ORFx in all isolates to
date
14
SCCmec elements are flanked on either end with
direct repeat (DRSCC) and inverted repeat
sequences (IRSCC)
DRSCC-L IRSCC-L
IRSCC-R DRSCC-R
15
Mec complex classes Trends Microbiol. 2001
Oct9(10)486-93Hiramatsu K, Cui L, Kuroda M,
Ito T.
In SCCmec type
II, III
N315
I, IV
V, VT
16
Association of PVL and SCCmec IV in different
genetic lineages
SCCmec II
SCCmec IV
ST5 ST1 ST8
ST30
17
Origin of SCCmecNon-mec SCC elements

• SCCcap1
• SCC 12263 (found in S. hominis GIFU12263)
• SCC- Composite Island (CI) (S. epidermidis)
• SCCpbp4 (S epidermidis within SCC CI)
• SCC fusidic acid resistance
• ACME in USA 300 and S. epidermidis

• IE25923- a 6 kb primordial non-mec cassette in
  MSSA strain,
• also in SCCmec IVc

A mobile genetic cassette for carrying cell wall
associated genes
18
Coagulase - staphylococci reservoirs for SCCmec
element diversity
19
Panton-Valentine leukocidin

• Panton, P. N., and F. C. O. Valentine. 1932.
  Staphylococcal toxin. Lancet 222506508.
• Present in (Prevost et al)
• Association w skin and soft tissue infections
  (Lina et al) regardless of MSSA in MRSA and
  necrotizing pneumonia (Lina et al and Gillet et
  al)

20
Panton-Valentine leukocidin toxin

• Cytolytic towards human and rabbit PMNs,
  macrophages,
• Associated with skin and soft tissue infections
  in CA-MSSA
• Now associated with CA-MRSA

21
PVL association w CA-MRSA

• Not found in HA-MRSA strains with SCCmec I, II,
  III
• More than 90 of CA-MRSA disease-causing strains
  bear pvl genes
• Has been present in all cases of severe sepsis,
  necrotizing fasciitis or nec pneumonia regardless
  of ST or presence of mecA gene
• (tightly associated w SCC mec IV and now SCCmec
  VT)
• Carried on a lysogenic phage integrated in the S.
  aureus genome

22
S. aureus has 6 cytolytic toxins

• Single component hemolysins
• a-hemolysin- Hla
• b-hemolysin- Hlb
• d-hemolysin- Hld
• Bicomponent toxins
• g-hemolysin- Hlg Hlg1 and Hlg2
• Leukocidin- Luk LukF and LukS
• Panton-Valentine Leukocidin LukS-PV and
  LukF-PV

23
Genetic organization of gamma toxin (Hlg), LUK
and PVL homologues
Hlg
DN
S F
dermonecrotic
LUK
hlg2
S S F
lukS PV
lukF PV
hlgA
ATCC49775
hlg2
lukM
P83
hlg2
lukE lukD
Newman
lukEv lukDv
hlg2
V8
hlg2
lukS-I lukF-I
S intermedius
6 combinations of proteins are possible within a
given strain
24
The S. aureus bicomponent toxinssynergohymenotro
phic (SHT)

• Two components assemble into a pore forming
  heteromultimer, F and S
• Each component is water soluble as a monomer
• Class F proteins
• LukFHlg1
• LukF-PV
• Class S proteins Hlg2, LukS, LukS-PV
• cell specificity
• phosphorylation by host protein kinase required

25
phiSLT, phiPVLcarry lukF-PV, lukS-PV

• ????????????2???????????????????????????????????
  ????????????????????????????????????????????2?????
  ???????2??????????????????????????????????????????
  Panton-Valentine???????(PVL)??????????????????????
  ?????????????????????? ??????????2????????????????
  ?????PVL??????????????????PVL??????????????fPVL???
  ??????????????????????????????PVL???????fSLT?????P
  VL???????PVL??????????????????????????????????????
  ????????????????PVL????????PVL????????????????????
  ???PVL???????PVL?????????????????????????????

26
Model for evolution of PVL toxin producing
community-acquired MRSA
MSSA
chromosome
27
Association of PVL and SCCmec IV together in
different genetic lineages
SCCmec II
SCCmec IV
Uniformly PVL-
PVL PVL-
PVL PVL
ST5 ST1 ST8
ST30
28
Recent Data from our group
29
CA-MRSA isolates from children tend to be more
susceptible than those from adults and
hospitalized children
David et al, PIDJ
Percent of Multidrug Resistant MRSA in Community-
and Hospital-
Onset Isolates, Comparing Pediatric and Adult
Samples
80.0
76.0
p 0.3520
66.7
70.0
p 60.0
52.3
50.0
40.0
Percent of Isolates
30.0
20.0
6.4
10.0
0.0
Adult
Pediatric
Adult Hospital-
Pediatric
Community-
Community-
Onset
Hospital-Onset
Onset
Onset
30
David et al.,
31
A Novel CA-MRSA Clone in Taiwan shifts the
paradigm

• Multiply resistant to non-b-lactams
• ST 59 (USA 1000)
• ALL SSTI disease isolates had PVL
• But few of the colonization isolates carried PVL
• Only 3 of 17 SSTI isolates carried SCCmec IV
• A majority were SCCmec nontypeable
• Sequencing led to type VT

32
Multiply resistant CA-MRSA strains from Taipei,
Taiwan J Clin Microbiology -Boyle-Vavra et al.
2005
!

• Disease (SSTI) vs colonization isolates both
  were usually multiply resistant

33
SCCmec VT more common than SCCmec IV among SSTI
isolates in Taipei
VT
VT
34
TaiwanSkin and Soft Tissue Infectionis
comprised of mainly PVL isolatesSCC mecVT
isolates
PVL
35
disease vs colonization Taipei
SSTI Colonization
T
T
pvl was present in all SSTIs, regardless of
SCCmec type. Most colonization isolates were
PVL- (SCCmecIV) PVL colonization isolates
associated w SCCmecVT
36
SCC mec VII? both ccr5 and ccr2 and mec class B.
SCCmec IV/V hybrid? Is this THE source of ccr5?
Controls
NEW

5 2
1 2 3 5
ccrC type
37
What role does PVL have in pathogenesis
38
Severe necrotizing disease (tip of the pyramid)
PVL
Skin and soft tissue
PVL
(PVL-)
colonization
39
CA-MRSA and CA-MSSA
petechial and desquamating rash of CA-MRSA and
MSSA
Adem et al. NEJM 35312 September 22, 2005
40
adrenals
lungs
Necrotizing pneumonia
bilateral adrenal hemorrhage
microabscess
Staph colonies
hemorrhagic infarction
Alveolar Architecture destroyed
Adem et al. NEJM 35312 September 22, 2005
41
Pulsed field gel of severe sepsis isolates
Genotype (type G), MLST 1, PFT USA 400
42
MLST(genotype)
43
Several genotypes have been there all along in
Chicago with USA 400 predominating among severe
sepsis patients
PVL genes
USA 400
PVL interpretation
USA 100
USA 300
WIS
44

• USA 400 (ST 1) was the first clone noted to cause
  severe sepsis and necrotizing pneumonia in
  Chicago and Minnesota.
• USA 300 was already present in a few CA-MRSA
  strains in Chicago but not in the most severe
  disease
• USA 300 (ST 8) has now taken over in Chicago
  (unpublished data), Atlanta, CA, and around the
  country
• Associated with necrotizing fasciitis and skin
  and soft tissue infections

45
Genome sequence of USA 3005 genetic islands
novel allotypes

• SCCmec IV - almost identical to that in MW2 (ST
  1)
• ?SA2usa PVL phage
• ?SA2usa
• Sak- fibrin specific blood clot dissolving enzyme
• Chp-chemotaxis inhibitory protein
• SaPI5-
• seq2, sek2
• ACME- encodes novel arginine deiminase and opp
• Also present in S epidermidis

46
Model I How PVL leads to tissue necrosis
PVL and other hemolysins secreted by
Staphylococcus aureus damage the membranes of
neutrophils, our first line of defense
Osmotic lysis releases Inflammatory and
cytotoxic compounds
PVL
neutrophil Lysis
neutrophils
Tissue necrosis Severe sepsis
47
paradox

• Patients with severe sepsis are leukopenic and
  neutropenic
• Yet they have massive inflammation of PMNs in
  necrotic tissue where bacterial abscesses are
  present
• If PVL lyses PMNs, why so many PMNS there?
• Perhaps macrophages also undergo apoptosis
  preventing clearance of neutrophils.

48
Model II Apoptosis

• direct interaction of PVL with mitochondrial
  membrane leads to release of cytochrome C which
  induces caspase 9 and eventual cell death
• Cytochrome c, with the help of a another
  molecule, Apoptosis Activation Factor-1 (Apaf-1),
  activates the caspases

49
Model for PMN apoptosis induced by low
concentrations of PVL
PVL
PVL receptor
Fatty acids, protein?
DNA fragmentation
Casapse 3
Caspase 9
50
activated after release of compounds from
mitochondrion
death signal from cell membrane
9
8
3
Nuclear destruction
51
rPVL localized to mitochondria during PVL induced
apoptosis
TEM ,5nM biotinylated PVL, 5 minutes, sagold
52
localization of PVL in PMNs rPVL in mitochondria
during PVL-induced apoptosis
Trate PMNS, Isolate mitochondrial fractions
LukS
( pores in mitochondria)
(no mito contam in cytososl)
Indicated no contamination of cytosol)
53
LukS protein seen in Lung patients died of
necrotizing pneumonia
Genestier et al. 2005
STAINED ANTI LUKs-pv MAB
54
Genestier et al., J Clin Invest. 115 2005

• Model II Apoptosis
• PVL at low concentrations (5 nM) induces
  apoptosis of PMNs eventually leading to PMN
  necrosis.
• Apoptosis occurs via a mitochondrial pathway
  involving Caspase 9
• But the mitochondrial pathway normally involves
  recruitment of Bax to the mitochondrial membrane
• But PVL itself appeared to be responsible for the
  death of the mitochindria possibly by forming
  pores in the mitochondrial membrane

55
We find wide distribution of PVL gene expression
levels among strains
Real time PCR assay using lukF specific probe
A
Unpublished data Boyle-Vavra, Malinis and Daum
56
Consequences of low vs high pvl gene expression

• LOW AMOUNTS INDUCE APOPTOSIS INSTEAD OF NECROSIS
  (Genestier et al)
• Significance unclear
• High amounts induce apoptosis
• Low amounts induce chemotactic mediators such as
  the release of leukotriene B4, interleukin 8

57
Bacteremic model of CA-MRSA disease

• Voyich et al J Immunol, 2005
• Tested two USA 400 strains (MR ands MS) and a USA
  300 strain (LAC)
• Demonstrated that 3 CA strains were more
  virulent than COL or MRSA 252.
• They caused more PMN lysis, invaded tissues
  better, and better survived PMN phagocytosis.
• Also there were greater numbers of USA 400
  strains recovered from lung, consistent with
  their involvement in nec pneumonia.
• Said Salim et al failed to show a difference in
  PMN lysis between PVL and PVL- strains however,
  they did not test ST1 isolates and might have
  chosen to test the lowest PVL expressors.

58
PVL unsolved mysteries

• Why is it associated with CA-MRSA?
• Why is it only associated with SCCmec IV or
  SCCmec VT?
• What selective advantage might it have in the
  community?
• Promoting spread due to promotion of inflammation
  and production of open sores.
• Nasal carriage? Probably not
• Skin carriage? Not well tested
• Where did it come from?
• Why is it more prevalent in MRSA than MSSA

59
Acknowledgements

• University of Chicago
• Robert Daum
• Shaohui Yin
• Kanok Mongkolrattanothai (now at Peoria)
• Daniel Kim
• Jie Peng
• Ben Ereshefsky
• Andrea Malinis
• Keiichi Hiramatsu, Juntendo
• Teruyo Ito, Juntendo
• Chih-Chien Wang, Tri-Services General Hospital,
  Taipei
• Linda Mac Dougal and Fred Tenover, CDC

60
Conclusion

• The high association of PVL with CA-MRSA might be
  due to the fact that most studies have been
  conducted on sick people. PVL is not essential
  for spread.
• Thus PVL may be more associated with CAMRSA
  disease than CAMRSA colonization, suggesting that
  PVL is important in the pathogenesis