1. METHICILLIN RESISTANT
STAPHYLOCOCCUS AUREUS
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2. What is MRSA?
• Methicillin Resistant Staphylococcus aureus
demonstrates resistance to semisynthetic
penicillins – Methicillin, Cloxacillin & Oxacillin.
• MRSA also exhibit resistance to
cephalosporins, monobactams and
carbepenamases
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3. • Hospital associated MRSA (HA-MRSA)  MRSA strains
that circulate and are transmitted to individuals in
health care facilities
• Risk factors for HA-MRSA
– Isolation of MRSA after 48hours of hospital admission
– History of Hospitalization, Surgery, Dialysis
– History of long term admission within one year of the
MRSA culture date
– Presence of indwelling catheter or percutaneous device at
the time of culture or previous to isolation of MRSA
• Community associated MRSA (CA-MRSA) 
– MRSA isolates obtained from individuals in community
who have not had recent exposure to the health care
system
– Patients in health care facilities in whom the infection was
present or incubating at the time of admission
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4. Methicillin / Meticillin
• Narrow spectrum antibiotic of penicillin class
introduced in 1959
• Has ortho-dimethoxy phenyl group attached
to side chain carbonyl group of penicillin
nucleus.
• Has been renamed as meticillin to have a
common international nonproprietary name.
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5. PENICILLIN METHICILLIN
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7. Why MRSA?
• Methicillin was initially used to detect
resistance to beta lactamase stable penicillins
hence the name MRSA.
• Oxacillin is used as alternative to methicillin
 ORSA (oxacillin resistant Staphylococcus
aureus)
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8. Mechanism of resistance to penicillins
• Presence of mecA gene
– mecA gene is carried on
Staphylococcal cassette
chromosome mec (SCCmec)
– SCC have mecA and ccr
(cassette chromosome
recombinase) and attach it
to the 3’ end of
staphylococcal chromosome
– Codes for modified penicillin
binding protein 2a (PBP 2a
OR PBP 2’)
– Has low affinity for Beta
lactams
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9. Mechanism of resistance to penicillins
• Production of Beta lactamase enzyme
– Some strains produce excessive Beta lactamase which makes it
appear borderline resistant to oxacillin (BORSA). Difficult to detect.
– MRSA with minor alterations to existing PBP  Moderately
resistant Staphylococcus aureus (MODSA)
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10. Virulence factors of Staphylococcus aureus
• Capsular polysacchrides • Hemolysins
• Peptidoglycan and Teichoic acids – Alpha Hemolysin
• Protein A – Beta Hemolysin
• Enzymes – Gamma Hemolysin
– Catalase – Delta Hemolysin
– Clumping factor – Panton-Valentine Leukocidin
– Coagulase • Toxins
– Fibrinolysins – Epidermolytic toxins
– Hyaluronidase – Enterotoxins A,B,C,D,E,H & I
– Lipases • Superantigens
– Phosphatidylinositol specific – Toxic shock syndrome toxin I
phospholipase C
– Nuclease
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11. Virulence Factors in CA-MRSA
Category Toxin
Cytolytic toxins Panton-Valentine leukocidin S and F
Fibronectin binding proteins A and B
Leukocidin R
Superantigenic toxins Enterotoxins (A to J)
Epidermolytic toxins
Toxic shock syndrome toxin -1
Enhanced growth and survival Arginine catabolic mobile element
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12. Nomenclature of MRSA
US pulse field Canadian Multi locus SCCmec type PVL presence
gel MRSA sequence
electrophoresi typing
s
USA 100 2 5 II NEGATIVE
USA 200 4 36 II NEGATIVE
USA 300 10 8 IVa POSITIVE
USA 400 7 1 IVa POSITIVE
USA 500 5,9 8 IV NEGATIVE
USA 600 1 45 II NEGATIVE
USA 700 72 IVa NEGATIVE
USA 800 2 5 IV NEGATIVE
USA 1000 59 IV POSITIVE
USA1100 30 IV POSITIVE
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13. Detection of MRSA
• Phenotypic detection systems
– Disc diffusion test
• Colony suspension prepared from 5colonies and plated on muller
hinton agar containing 2-4% NaCl at neutral pH.
• Oxacillin disc (1ug) placed and incubated at 35°C for 24 hours
• <10mm is considered resistant, >13mm is considered sensitive.
• For isolates with intermediate results
– Test for mec A, PBP2a
– Cefoxitin disc test
– Oxacillin MIC test or
– Oxacillin salt agar screen test may be performed.
• Any growth within the zone of inhibition indicates oxacillin
resistance
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14. Other detection methods
• Agar dilution test
– 0.5 McFarland preparation of isolate is spot inoculated on MHA with
2% NaCl containing 256 – 0.125 ug oxacillin/ml in serial doubling
dilutions
– MIC of >4ug/ml is considered resistant , MIC <2ug/ml is considered
susceptible
• Broth microdilution
– Muller hinton broth inoculated with inoculum density of 5 X 10 5 cfu/ml
• Breakpoint methods
– Includes both agar and broth methods but test only the breakpoint
concentration (2mg/L oxacillin, 4mg/L methicillin)
• E-test oxacillin MIC test
– Easy to set up as a disc diffusion test
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15. • Oxacillin screen agar
– MHA with 4%NaCl and 6ug/ml oxacillin
– Inoculated with 10uL of 0.5Mcfarland preparation, streaked in one
quadrant and incubated at 35°C for 24-48 hours
– Any growth after 24hrs is considered oxacillin resistant
• Cefoxitin disc diffusion test
– Cefoxitin – potent inducerof mecA regulatory system
– Used as a surrogate marker for detection of mecA gene mediated
methicillin resistance
– Inducible resistance to methicillin is better seen with cefoxitin than
oxacillin. This is due to enhanced induction of PBP 2a by cefoxitin.
– 30ug cefoxitin disc is used.( < 21mm is resistant and > 22mm
susceptible)
• PBP 2a latex agglutination kit
– PBP2a extacted from suspension of colonies and react with latex
particles coated with monoclonal antibody against PBP 2a
– Visible agglutination indicates positive result and presence of PBP 2a
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16. Chromogenic Screening medium
Media Components/ action Colony colour
CHROM ID CEFOXITIN 4MG/L GREEN
TARGETS ALPHA
GLUCOSIDASE
ORSAB MANNITOL SALT AGAR BLUE
OXACILLIN 2MG/L
POLYMYXIN
ANILINE BLUE
MRSA INDENT AGAR CEFOXITIN RUBY COLOURED
CHROMOGENIC
PHOSPHATASE SUBSTRATE
DENIM BLUE AGAR CEFOXITIN DENIM BLUE COLONIES
PHOPHATIDASE ACTIVITY
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17. • Molecular methods
– Detection of mecA gene by PCR is considered gold
standard
• DNA extraction
• mecA gene amplified using specific primers
– 30 cycles of denaturation at 94°C for 45seconds
– Anneling at 50°C for 45 seconds
– Extension at 72°C for 1 minute
– Final extension step at 72°C for 3mins
• PCR products visualized on 2%agarose gel with
ethidium bromide dye under U-V transluminator
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18. Dr.Riyaz Sheriff 18

19. Community associated meticillin-
resistant Staphylococcus aureus
strains as a cause of healthcare-
associated infection
J.A Otter, G.L. French
Journal Of Hospital Infection 79
(2011) 189-193
Impact factor 3.393
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20. Introduction
• CA-MRSA infections were first reported in early
1990s from western Australia, New Zealand and
in America.
• CA-MRSA seems to have evolved from MSSA
acquiring SCCmec cassettes.
• Have the ability to affect younger, healthy people
and spread rapidly in community settings.
• CA-MRSA are generally susceptible to non Beta
Lactam antibiotics, Have small SCCmec cassettes
(type IV or V), Many of them produce PVL
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22. CA-MRSA in Hospital outbreaks
• Nosocomial outbreaks reported from 2003 from North
America, Germany, Israel, Switzerland, Greece and UK.
• Most outbreaks have been caused by single cross
infecting strain
• Infections in health care workers and transmission to
their household contacts have occurred in several of
these outbreaks
• PVL + CA-MRSA has been isolated in most of the
outbreaks but one outbreak in Israel and two
outbreaks in UK were caused by PVL negative strains
 CA-MRSA do not need PVL to cause nosocomial
infections
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23. Control Measures for CA-MRSA
• Isolation of affected patients
• Screening other patients in the unit for asymptomatic
carriage
• Reinforcement of standard infection control
procedures
– Hand hygiene
– Screening of staff members for colonization
– Swabbing of environmental surfaces and equipments
– Improved environmental cleaning
• Closure of unit to new admissions
• Screening of household contacts of health care
workers and appropriate management during CA-
MRSA outbreaks
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24. CA-MRSA role in endemic HAI
CA-MRSA is overtaking HA-MRSA as a cause of HAI
Initially CA-MRSA was reported in new born infections
and post operative prosthetic joint infections
Recent reports suggest CA-MRSA as a cause of HA
bacteraemias.
16% of hospital onset infections and 22% of health
care associated infections are caused by CA-MRSA
Many countries have reported 10fold increase in CA-
MRSA from 1999 to 2006
In Greece PVL positive CA-MRSA accounted for 45% of
HA-MRSA infections at several hospitals during 2001-
2003
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25. CA-MRSA implications
 CA-MRSA is emerging as a cause of hospital associated infections in Asia,
Africa & South America. The reason for this emergence is not clearly
understood.
 CA-MRSA have the ability to affect healthy individuals
 CA-MRSA strain with PVL are on the rise resulting in higher virulence.
 Studies have shown that CA-MRSA behave like HA-MRSA once inside the
hospitals but cause more invasive infections than uncomplicated SSTI.
 CA-MRSA exposed to antibiotics is more likely to acquire resistance genes
and become MDR strains like HA-MRSA
 Definitions for HA-MRSA and CA-MRSA become more confused.
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26. CA-MRSA implications (Cont’d)
 Control of MRSA in hospital settings will be more difficult.
 With lack of clear knowledge on epidemiology of CA-MRSA the current
infection control policies may not be successful and needs to be
modified.
 Hospitals should type MRSA strains involved in outbreaks and there
should also be periodic assessment of the antibiotic resistance profiles
among CA-MRSA strains.
 The success of CA-MRSA in spreading in various subsets of the
community is a major concern.
 Finally there is an urgent need to measure the prevalence and
epidemiology of CA-MRSA and also develop systems to identify and
control these organisms in the community, hospitals and other health
care facilities.
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27. Conclusion
 CA-MRSA has emerged as a Nosocomial pathogen in the recent years and
are beginning to overtake HA-MRSA in hospital infection.
 CA-MRSA infections puts a wider group of people at risk. This includes
hospitalized patients, Health workers and their community contacts
 Classification of CA-MRSA and HA-MRSA is becoming more confusing
 Western countries report a MRSA incidence ranging from 10.4% to 40%
 In Tamil Nadu MRSA prevalence in clinical samples is estimated to be
around 31.1% in clinical and 37.9% in carrier samples
 MDR among MRSA in clinical isolates was estimated around 63.6% and
23% carrier samples
 There is an urgent need to measure the prevalence and epidemiology of
CA-MRSA and also develop systems to identify and control these
organisms in the community, hospitals and other health care facilities.
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28. References
• Koneman’s atlas and textbook of diagnostic microbiology
• Essentials of medical pharmacology KD tripathi 5th edition
• Michelle barton et al Guidelines for prevention and management of
community - associated methicillin - resistant Staphylococcus
aureus: a perspective for Canadian health care practitioners,
Canadian journal of infectious disease and medical microbiology vol
17, supplement C 2006 page 4C-24C
• Rajadurapandi et al prevalence and antimicrobial susceptibility
pattern of methicillin resistant Staphylococcus aureus: a
multicentre study, IJMM (2006) 24 (1): 34-38
• Benjamin etal Reduced Vancomycin Susceptibility in Staphylococcus
aureus, Including Vancomycin-Intermediate and Heterogeneous
Vancomycin- Intermediate Strains: Resistance Mechanisms,
Laboratory Detection, and Clinical Implications, CMR 2010 p.99-139
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