1. Methicillin-Resistant Staphylococcus aureus in Community Settings
Ruth Lynfield, M.D.
Minnesota Department of Health

2. Emergence of Resistance in S. aureus
Time required for prevalence rates of resistance to reach 25% in hospitals
Drug
Year Drug Introduced
Years to Report of Resistance
Years Until 25% Rate in Hospitals
Years Until 25% Rate in Community
Penicillin
1941
1-2 6
15-20
Methicillin
1961
<1
25-30
40-50 (projected)
Chambers, EID 7: , 2001

3. Methicillin Resistance in S. aureus
mecA confers resistance to penicillins and cephalosporins
mecA is part of the staphylococcal cassette chromosome mec (SCCmec), a mobile genetic element (21-67 kb in size) that may also contain genes that confer resistance to non-beta-lactam antimicrobials
SCCmec has been classified into 5 main types based on polymorphisms in its conserved genes

4. Staphylococcal Cassette Chromosome mec Types
SCCmec type I - Archaic clone LJ RJ
D mecR1
pls
HVR
dcs
orfX
ccrAB1
IS1272
SCCmec type IA - Iberian clone LJ RJ
DmecR1
DHVR
dcs
orfX
pls
ccrAB1
IS1272
pUB110
SCCmec type III - Hungarian clone LJ
ips RJ
YccrAB
orfX
YTn554
HVR
pT181
pI258
Tn554
ccrAB3
mecI
mecR1
SCCmec type IIIA - Brazilian clone LJ RJ
YccrAB
orfX
YTn554
mecI
mecR1
HVR
pI258
Tn554
ccrAB3
SCCmec type II - NY/Japan clone
10 Kb LJ RJ
dcs
orfX
kdp
ccrAB2
Tn554
mecR1
mecI
mecR1
mecI
HVR
HVR
pUB110
mecA
IS431
SCCmec type IV - Pediatric clone LJ RJ
DmecR1
dcs
orfX
LJ - chromosomal left junction
RJ - chromosomal right junction
ccrAB4
IS1272
HVR
Oliveira et al., Lancet ID, 2002

5. Background: Healthcare-associated (HA) MRSA
Leading cause of nosocomial pneumonia, surgical wound infection, and bloodstream infection
Established risk factors include
Current or recent hospitalization
Residence in long-term care facilities
Dialysis
Typical resistance profile
Resistant to many antimicrobials in addition to beta-lactams

6. Community-Associated Methicillin-Resistant Staphylococcus aureus (CA-MRSA)
Reports began in 1980s of MRSA occurring in the community in patients without established risk factors
Younger patients
Indigenous peoples and racial minorities
Skin infections common
Outbreaks:
Injection drug users
Players of close-contact sports
Prison/jail inmates
Group Homes (developmentally disabled)
Men who have sex with men
Military recruits

7. CA-MRSA
Isolates typically susceptible to most antimicrobial classes other than beta-lactams
Isolates differ by PFGE from HA-MRSA
Isolates have different SCCmec types from HA-MRSA
Isolates have been identified that are highly related to MSSA except for SCCmec element*
Although most infections associated with CA-MRSA have been skin and soft tissue, some infections have been very severe including necrotizing pneumonia and other life-threatening infections
* Fey. Antimic Agents Chem. 2003; 47:
Mongkolrattanothai. Clin Infec Dis. 2003: 37:

8. CA-MRSA in Minnesota
1997- Minnesota Department of Health (MDH) received reports of MRSA infections in young, previously healthy individuals
Reported four pediatric deaths due to MRSA infection (MMWR, August 20, 1999)

9. Four Pediatric Deaths, CA-MRSA Minnesota and North Dakota, 1997-99
Clonal group A
Clonal group A (MW2)
PFGE
T/S, tet, cip, gent, ery, clind, vanc
Antimicrobial
susceptibility
Necrotizing pneumonia/ sepsis
Sepsis
Septic joint, pneumonia/ empyema
Syndrome
White
American Indian
African American
Race
Male
Female
Gender
12 months
13 years
16 months
7 years
Age
Case 4
Case 3
Case 2
Case 1

10. CA-MRSA in Minnesota
Minnesota Communicable Disease Reporting rule amended in 1999:
All cases of serious illness or death due to CA-MRSA reportable
Sentinel sites were required to report all cases of MRSA

11. Minnesota Definition of CA-MRSA Used in Prospective Surveillance
Positive culture for MRSA obtained within 48 hours of admission (if hospitalized)
No history of hospitalization in past year
No history of surgery in past year
No history of long-term care in past year
No history of dialysis in past year
No permanent indwelling catheters or percutaneous medical devices
No prior history of MRSA infection or colonization

12. Prospective MRSA Surveillance: Methods, MN, 2000
12 sentinel hospitals selected to represent different geographic regions (6 metro area, 6 greater MN) reported all cases MRSA
Patient information was collected and MRSA isolates were obtained for all cases (HA and CA-MRSA)
Presumptive CA-MRSA patients were interviewed to verify that they met the CA-MRSA case definition

13. Minnesota MRSA Surveillance
MRSA patients
Risk factors (+)
Healthcare-associated
Hospital-acquired
Community-onset with Risk factors
Medical record review
Risk factors (-)
Community-onset, no risk factors documented in medical record
Telephone interview
Indeterminate
Risk factors (+)
No interview
Risk factors (-)
Community-associated

14. Minnesota MRSA Surveillance Methods: Laboratory
MRSA isolates from sentinel sites sent to MDH Laboratory
All CA-MRSA isolates tested
25% of HA-MRSA isolates from each site randomly selected for testing
Isolates confirmed as S. aureus (tube coagulase)
Antimicrobial susceptibility testing (including oxacillin) by broth micro-dilution
PFGE subtyping
26 CA and 26 HA-MRSA isolates characterized for toxins

15. Minnesota MRSA Surveillance Results: 2000
4,612 patients with S. aureus identified at 10 sentinel sites in 2000 (total number S. aureus unavailable at two sites)
1100 (25%) were MRSA (range 10-49%)
937 (85%) were HA-MRSA
131 (12%) were CA-MRSA (range 4-50%) after patient interview*
32 (3%) not enough information to classify
*13% of presumptive CA-MRSA cases were reclassified as HA-MRSA after interview

16. Age and Culture Sites of CA-MRSA Cases, MN, 2000
HA-MRSA
(n = 937)
p-value
Age (median)
23 years
68 years
<0.05
Culture site
No. (%)
Skin
98 (75)
343 (36)
Respiratory
8 ( 6)
205 (22)
Blood
5 ( 4)
83 ( 9) NS
Urine
1 ( 1)
185 (20)
Other
10 (7)
110 (12)

17. CA and HA-MRSA PFGE Subtype Patterns, Minnesota, 2000
Healthcare-associated (clonal group H)
Community-associated (clonal group A)

18. Relatedness to Reference Strain
PFGE Relatedness of CA and HA-MRSA Isolates to Reference Strain, MN, 2000
Relatedness to Reference Strain
100%
90%
70%
50%
35%
15%
130
120
Clonal Group A
110
Community-associated (n=106)
Healthcare-associated (n=211)
100 90 80 70
Number of Isolates 60 50 40 30 20 10 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
(MW2)
Number of Bands Different from MRSA Reference Strain

19. Initial Empiric Treatment of CA-MRSA Infections, MN, 2000 (n = 92)

20. Susceptibility of CA and HA-MRSA Isolates by Antimicrobial Agent, MN, 2000
100
100 95 96 94 94 92 92 90 83 79 80 44 21 16 9
p<0.001
p<0.001
p<0.001
p=0.001

21. Susceptibility of CA-MRSA Isolates by Year, MN 1996-2003
x2=5.29
p=0.02
x2=13.97
p<0.001
x2=59.22
p<0.001

22. Macrolide Resistance Mechanisms
in S. aureus
Macrolides (e.g., erythromycin)
Lincosamides (e.g., clindamycin)
Streptogramin B
Protein
synthesis
erm
msrA
Ribosome
Efflux pump
Methylase
All MLS antibiotics bind basically the same target site on the ribosome in the bacterial cell. This inhibits protein synthesis.
Resistance in staph in encoded by two major mechanisms:
erm (erythromycin resistance/ribosome mechanism) gene: this gene encodes a methylase that affects the target binding site on the ribosome. Methylase is an enzyme that adds a methyl group to the ribosome which methylates the binding site and blocks the binding of all MLS antibiotics.
msrA (methionine sulfoxide reductase) gene: this gene encodes an efflux pump which pumps macrolides (only) out the the bacterial cell before they can bind to the ribosome target site. Bacteria will still be susceptible to lincosamides (clindamycin) and streptogramin B (synercid is a comination of streptogramin A and B)
Macrolides
Lincosamides
Streptogramin B
Macrolides

23. Regulation of erm Methylase
Macrolides
Constitutive
Inducible
ermC
ermI
Methylase
Methylase
Methylase
Regulation of erm methylase:
The erm gene can be regulated in two ways. Expression of resistance can be constitutive (turned on all of the time) or inducible meaning it is turned on (producing methylase) only in the presence of a macrolide inducer.
When a macrolide enters the bacterial cell, the erm gene turns on the the staph strain will be resistant to macrolides. The strain will be phenotypically susceptible to lincosamides and streptogramin B, but will have genotypical potential for resistance.

24. Inducible Macrolide-Lincosamide-Streptogramin B (MLSB) Phenotype
Zone of Inhibition
Typically, disk diffusion is done with the erythromycin and clindamycin disks placed at some distance apart on the plate. However, if the two disks are placed more closely together the erythromycin will diffuse into the clindamycin zone of inhibition and turn on the erm gene. We will then see a blunting of the effect of clindamycin on the staph – the organism will grow up to the disk on the side near the erythromycin disk. E C
Blunted Zone

25. Clindamycin Resistance, CA-MRSA Isolates, MN, 2000-2001
60% of erythromycin-resistant isolates were susceptible to clindamycin by broth microdilution
Erythromycin-resistant, clindamycin-susceptible isolates were tested by double disk diffusion to evaluate for inducible resistance
84% had inducible clindamycin resistance, all contained the erm gene (45 ermC, 7 ermA, 2 ermC and msrA, 3 ermA and msrA)
16% did not have inducible clindamycin resistance and only contained the msrA gene (11 isolates)

26. Clindamycin Resistance, CA-MRSA Isolates, MN, 2002-2003
76% of erythromycin-resistant isolates were susceptible to clindamycin by broth microdilution
Erythromycin-resistant, clindamycin-susceptible isolates were tested by double disk diffusion to evaluate for inducible resistance
42% had inducible clindamycin resistance
Overall between , 51% of erythromycin resistant, clindamycin susceptible isolates by broth microdilution, had inducible clindamycin resistance (x2 trend from = 43.7, p<0.001)

27. Clinical Implications
Constitutive mutants, which can occur at a frequency of ~10-7cfu, may be selected during a course of clindamycin therapy in patients with infections due to inducible clindamycin-resistant strains of S. aureus

28. Genome and Virulence of CA-MRSA
CA-MRSA isolate from one fatal case sent to Japan (PFGE subtype MR 14A)
CA-MRSA (MW2) genome sequenced
MW2 grows faster than HA-MRSA strains
MW2 has SCCmec type IVa (types I-III typically seen in HA-MRSA strains)
MW2 has unique virulence genes (including PVL)
Lethal dose of MW2 in an animal model is five times less than a comparison Japanese HA-MRSA strain
Baba et al. Lancet. 2002; 359:

29. Panton-Valentine Leukocidin (PVL)
S. aureus cytotoxin formed from 2 synergistic proteins
Causes tissue necrosis (skin and mucosa) and leukocyte destruction by forming pores in cellular membranes
PVL identified in 7 isolates from fatal CA-MRSA MN patients; 6 with necrotizing pneumonia

30. Effects of PVL on PMNs
Without PVL
With PVL

31. Exotoxin Genes and Gene Alleles
26 CA-MRSA and 26 HA-MRSA isolates underwent polymerase chain reaction to detect gene sequences
Panton-Valentine leukocidin*, staphylococcal enterotoxins A,C, H, and K, accessory gene regulator 3 and SCCmec IV were significantly more likely to be found among CA-MRSA isolates
Staphylococcal enterotoxins D, G, I, J, M, N, O, accessory gene regulator 2, and SCCmec II were significantly more likely to be found among HA-MRSA isolates
* Produces exotoxin causing tissue necrosis and leukocyte destruction, has been associated with cases of necrotizing pneumonia, skin abscesses

32. Potential Prevalence of CA-MRSA, 2001 - 2002: GA, MN, MD
Healthcare-associated
Community-associated
Probable CA
Georgia
n=7819
Minnesota
n=3014
Maryland
n=1720
Regional differences in macrolide resistance and predominant clonal groups between GA and MN

33. Management of Skin and Soft Tissue CA-MRSA Abscesses
Observational study: 69 children, TX 5/02-3/03, ER/acute care clinic
Incision and drainage effective management (only 7% given initial antibiotic active against their isolate)
Predictor of hospitalization was initial lesion > 5 cm (p=0.004)
Lee. Ped Infec Dis J. 2004; 23:

34. Hospital Transmission of CA-MRSA
8 postpartum women developed skin and soft tissue infections
5 re-hospitalizations, 3 required surgical drainage
Isolates indistinguishable by PFGE
CA-MRSA strain (MW2)
Saiman. Clin Infect Dis. 2003; 37:

35. Conclusions
CA-MRSA strains have distinct molecular and microbiologic features compared with HA-MRSA strains
The emergence of MRSA in the general population has important clinical implications because empiric therapy is generally a beta-lactam antimicrobial agent
Additional evaluation needed on treatment options for non-severe infection
CA-MRSA susceptibility trends in other antimicrobial classes should be monitored

36. Conclusions (cont.)
More information is needed on the clinical impact of inducible clindamycin-resistant strains
More information is needed on the impact of bacterial virulence factors on severity of CA-MRSA infections
Risk factor studies are needed to identify persons at risk and optimal methods of prevention
CA-MRSA strains have been detected in hospital settings and have been associated with nosocomial infection

37. Antimicrobial Resistance
“… the microbes are educated to resist penicillin and a host of penicillin-fast organisms is bred out which can be passed on to other individuals and perhaps from there
to others until they reach someone
who gets a septicemia or a pneumonia which penicillin cannot save. In such cases the thoughtless person playing with penicillin treatment is morally responsible for the death of the man who finally succumbs to infection with the penicillin-resistant organism. I hope this evil can be averted.”
~ Sir Alexander Fleming, New York Times, 6/26/45

38. Acknowledgements
Active Bacterial Core Surveillance Staff, Infection Control Practitioners and Microbiologists in CT, GA, MD, MN; Monica Farley, Lee Harrison
CDC: Scott Fridkin, Jeff Hageman, Monina Klemens, Melissa Morrison and Fred Tenover
MDH: Joanne Bartkus, John Besser, Dave Boxrud, Jessica Buck, Elizabeth Cebelinski, Kathy Como-Sabetti, Richard Danila, Anita Glennen, Billie Juni, Kathy LeDell, Beth Shade,
Sarah Vetter