methicillin resistant staphylococcus aureus (mrsa… · 1 dtu food, technical university of denmark...
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November 2011 EURL course 1 DTU Food, Technical University of Denmark
Methicillin resistant Staphylococcus aureus (MRSA) Lina Cavaco [email protected]
November 2011 EURL course 2 DTU Food, Technical University of Denmark
Staphylococcus aureus • Gram positive cocci • Catalase positive • Coagulase postive • Inhabitants of skin and mucosa in animals and man • Circa 25-30% carriers of Staphylococcus aureus, less than 2% carry
MRSA – Principal carriage site- nasal cavity – Perineum – Axillae
• Oportunistic pathogens
November 2011 EURL course 3 DTU Food, Technical University of Denmark
Staphylococcus aureus as pathogen
• Humans –Skin infections –Wound infections –Soft tissue infections –Enterotoxinogenic strains- diarrhoea –Necrotizing pneumonia –Septicaemia
• Mostly Hospital acquired infections • Some community acquired infections (increasing trend)
November 2011 EURL course 4 DTU Food, Technical University of Denmark
Staphylococcus aureus as pathogen
• Animals –Mastitis in cows –Skin and soft tissue infections in pets –Skin infections in production animals –Emergence also of MRSP (Methicillin Resistant
Staphylococcus pseudointermedius)
November 2011 EURL course 5 DTU Food, Technical University of Denmark
Resistance in S. aureus
The first treatment: penicillin (1930); end 40’s: 50% S. aureus
resistant) 1959: introduction methicillin; after 3 months 3/5000
R 2009: high frequent resistance against penicillines (by
enzyme penicillinase/beta-lactamase) => clavulanic acid!)
Resistance against other groups of antimicrobials ‘’not
extreme’’
November 2011 EURL course 6 DTU Food, Technical University of Denmark
S. aureus becomes MRSA…..
• Methicillin resistant Staphylococcus aureus- derives from the first antistaphylococcal drug, methicillin and defines resistance to all beta-lactam drugs
• contains mecA gene in Staphyloccal Chromosome Cassette - SCCmec
• Resistant against all beta-lactam
antimicrobials -penicillines,
cephalosporines (beta-lactamase inhibitors like clavulanic acid are not
active)
November 2011 EURL course 7 DTU Food, Technical University of Denmark
Resistance mechanism • mecA target replacement
PBP2a – PBP2a has low affinity to
beta lactam drugs – Inibition of drug activity
• mecA located in the Staphylococcal cassette chromossome mec (SCCmec)
– Large genetic element – several different SCCmec
cassettes found in MRSA • A new mecA homologue
was found recently in a novel cassette called SCCmec XI
Chongtrakool et al. 2006
Chongtrakool et al., 2006
November 2011 EURL course 8 DTU Food, Technical University of Denmark
MRSA (definition of classification)
• Hospital acquired (HA)-MRSA – Most infections that become clinically
evident after 48 hours of hospitalization.
– Infections that occur after the patient is discharged from the hospital can be considered healthcare-associated if the organisms were acquired during the hospital stay.
– Causes bacteriæmia, pneumonia, surgery wound infections…
– Well characterized hospital clones harbouring large SCCmec cassettes (I-III)
November 2011 EURL course 9 DTU Food, Technical University of Denmark
MRSA (definition of classification)
• Community acquired (CA)-MRSA – Diagnosis of MRSA was made in the outpatient setting or by a culture
positive for MRSA within 48 hours after admission to the hospital.
• No medical history of MRSA infection or colonization. • No medical history in the past year of:
– Hospitalization – Admission to a nursing home, skilled nursing facility, or hospice – Dialysis – Surgery
• No permanent indwelling catheters or medical devices that pass through the skin into the body.
– Mostly causes skin and soft tissue infections, but can be more serious – Strains fit into diverse backgrounds harbouring small SCCmec
cassettes (IV and V) – Some contain associated virulence factors such as PVL
November 2011 EURL course 10 DTU Food, Technical University of Denmark
MRSA (definition of classification)
• LA-MRSA – Associated to clones adapted to
livestock (ST398, ST97) • Mostly pigs but other species found
as carriers (veal calfs, poultry, horses, pets)
– Carried by healthy and also sick animals
– Human carriage and /or infection associated to direct or indirect contact to animal sources
– Found in environmental samples (dust) in farms
– Found in meat, even though considered as low risk for food handlers and consumers
November 2011 EURL course 11 DTU Food, Technical University of Denmark
Epidemiology of MRSA in humans • Hospital-acquired infections
influenced by: – Distribution of HA-clones – Infection control measures – Antimicrobial usage – Search and destroy policies
• Community-acquired infections
– Sporadic cases but also some outbreaks
– Associated to specific clones (USA300) but also other sporadic clones
•Very different prevalences between EU countries
November 2011 EURL course 12 DTU Food, Technical University of Denmark
Epidemiology of MRSA in animals
• Pets and companion animals
– Most human strains – Sporadic cases – More problematic Vet
health issue: MRSP
• Cows: Mastitis
• Pigs, veal calves, poultry – ST398 main LA-MRSA clone – animal carriers, sporadic
infections – Widespread in Europe
http://www.efsa.europa.eu/en/efsajournal/doc/1376.pdf
Figure 7: Prevalence of MRSA positive production holdings,
MRSA EU baseline survey in breeding pigs, 2008(a)
November 2011 EURL course 13 DTU Food, Technical University of Denmark
Epidemiology of MRSA – Human clones
Companion animals
Transmission
Human carriers:
November 2011 EURL course 14 DTU Food, Technical University of Denmark
CC398 LA MRSA with broad host range
Contact with animals is main risk factor for humans
November 2011 EURL course 17 DTU Food, Technical University of Denmark
Potential Risk factors for CC398
•Vertical dissemination from breeding holdings
•Holding size •Trade •Environmental contamination
•Antimicrobial use •Selection/ co-selection
–Still lacking knowledge on how factors interplay!
November 2011 EURL course 18 DTU Food, Technical University of Denmark
New MRSA carrying the mecALGA251 gene
• Firstly found in the UK – Mastitis isolate – Phenotypic resistance – mecA not detected, PBP2a negative – Overexpressed betalactamase test negative – Full genome sequenced
1. Holmes MA, Zadoks RN. Methicillin Resistant S. aureus in Human and Bovine Mastitis. J Mammary Gland Biol Neoplasia. 2011 Oct 8. [Epub ahead of print]
2. García-Álvarez L, Holden MT, Lindsay H, Webb CR, Brown DF, Curran MD, Walpole E, Brooks K, Pickard DJ, Teale C, Parkhill J, Bentley SD, Edwards GF, Girvan EK, Kearns AM, Pichon B, Hill RL, Larsen AR, Skov RL, Peacock SJ, Maskell DJ, Holmes MA. Meticillin-resistant Staphylococcus aureus with a novel mecA homologue in human and bovine populations in the UK and Denmark: a descriptive study. Lancet Infect Dis. 2011 Aug;11(8):595-603.
3. Shore AC, Deasy EC, Slickers P, Brennan G, O'Connell B, Monecke S, Ehricht R, Coleman DC. Detection of staphylococcal cassette chromosome mec type XI carrying highly divergent mecA, mecI, mecR1, blaZ, and ccr genes in human clinical isolates of clonal complex 130 methicillin-resistant Staphylococcus aureus. Antimicrob Agents Chemother. 2011 Aug;55(8):3765-73.
4. Cuny C, Layer F, Strommenger B, Witte W. Rare Occurrence of Methicillin-Resistant Staphylococcus aureus CC130 with a Novel mecA Homologue in Humans in Germany. PLoS One. 2011;6(9):e24360.
SCCmec type XI containing divergent mecALGA251
November 2011 EURL course 19 DTU Food, Technical University of Denmark
New mecALGA251 gene distribution in the UK- human and animal isolates and their spa and CC types
García-Álvarez L, Holden MT, Lindsay H, Webb CR, Brown DF, Curran MD, Walpole E, Brooks K, Pickard DJ, Teale C, Parkhill J, Bentley SD, Edwards GF, Girvan EK, Kearns AM, Pichon B, Hill RL, Larsen AR, Skov RL, Peacock SJ, Maskell DJ, Holmes MA. Meticillin-resistant Staphylococcus aureus with a novel mecA homologue in human and bovine populations in the UK and Denmark: a descriptive study. Lancet Infect Dis. 2011 Aug;11(8):595-603.
November 2011 EURL course 20 DTU Food, Technical University of Denmark
So far the new mecALGA251 homologue has been found in animal and human isolates from:
Holmes MA, Zadoks RN. Methicillin Resistant S. aureus in Human and Bovine Mastitis.
J Mammary Gland Biol Neoplasia. 2011 Oct 8. [Epub ahead of print]
November 2011 EURL course 21 DTU Food, Technical University of Denmark
Detection of MRSA • Routine susceptibility testing
– Screening tests (oxacillin, cefoxitin) • Selective procedures for isolation • Confirmation of presence of resistance determinant
– Molecular detection of mecA (PCR) (different PCR primers for new homologue gene)
– Detection of PBP2a with imunoassays (rapid agglutination tests) (not suitable for detection of new PBP)
• The genotypic confirmation is compulsory for MRSA identification
November 2011 EURL course 22 DTU Food, Technical University of Denmark
Phenotypic detection • Oxacillin screening test –
CLSI guidelines describe procedure in Appendix B.
• Cefoxitin testing – Disk diffusion
• <=21mm
– Broth dilution • >4mg/L
• Due to new mecA
homologue genotypic detection by mecA PCR might no longer be sufficient
November 2011 EURL course 23 DTU Food, Technical University of Denmark
Selective isolation procedures (Baseline method)
• Selective isolation procedure using
– pre enrichment in Mueller Hinton Agar w 6,5%NaCl,
– enrichment in TSB with 3,5 mg/L cefoxitin and 75 mg/L aztreonam
– plating on Chromogenic Agar (Brilliance MRSA Agar or MRSA 2) or equivalent and on blood agar
– Isolation up to 5 colonies
November 2011 EURL course 24 DTU Food, Technical University of Denmark
Confirmation of id and methicillin resistance status- PCF we used until now
•PCR 16S, mecA and nuc for MRSA ID
–16S- confirms that the PCR works
–mecA – Confirm methicillin resistance
–Nuc- confirm ID (only positive in Staphylococcus aureus)
16S
mecA
nuclease
November 2011 EURL course 25 DTU Food, Technical University of Denmark
Need for new methods to detect new mecA homologue • With the possibility of this new gene, it is not sufficient ot test for
mecA! • Agglutination tests directed to PBP2a fail in detecting resistance • Hyperproduction of beta-lactamase not detected in these isolates
as cause of resistance • Phenotype more important for detection! • Need to implement new PCR methods (you will try one in this
course, which is still under implementation)
November 2011 EURL course 26 DTU Food, Technical University of Denmark
New mecA- need to change methods!! • PCR for mecA, spa, pvl and
mecALga251 will be used in the practical part of this course
– mecA – Confirm classic
methicillin resistance – mecALGA251 – new gene – Spa confirm ID (only positive
in Staphylococcus aureus) and used for spa typing directly
– PVL- Determination of the presence of the Panton Valentine Leucocidin gene
Published protocol from:
Larsen AR, Stegger M, Sørum M. spa typing directly from a mecA, spa and pvl multiplex PCR assay-a cost-effective improvement for methicillin-resistant Staphylococcus aureus surveillance. Clin Microbiol Infect. 2008 Jun;14(6):611-4.
with new update that will be published soon:
Additional amplification of mecALGA251 – 138bp
November 2011 EURL course 27 DTU Food, Technical University of Denmark
Typing of MRSA- PFGE
• Typing of strains –PFGE with SmaI most
frequently (except for ST398 which is not typable with PFGE with SmaI ) - very discriminatory method
• Subjective but very discriminatory, useful for outbreak investigation
Rotun et al., 1999.
November 2011 EURL course 28 DTU Food, Technical University of Denmark
Typing of isolates- spa- typing
•Typing of strains –Single-locus sequencing
• Spa-typing (Shopsin et al., 1999)
– Easy and
reproducible method with good correlation to MLST (CC), some exceptions
http://www.uniklinik-freiburg.de/iuk/live/molhyglabor/leistungskatalog/spa.jpg
November 2011 EURL course 29 DTU Food, Technical University of Denmark
Typing of isolates- MLST
• Multi locus sequence typing (MLST) based on sequencing 7 housekeeping genes and determination of their allelles (Enright et al., 2000)
– Large-scale
epidemiological linkage
November 2011 EURL course 30 DTU Food, Technical University of Denmark
Typing of isolates - SCCmec
•Typing of Staphylococcal chromossomal cassete mec (SCCmec)
–Typing of the SCC element responsible for resistance- • Origin of SCCmec • Longer trend epidemiology and evolution
– SCCmec acquisition into strain background
–SCCmec subtyping • Determine SCCmec subtypes • Direct repeat unit (DRU)- typing • ccrB and ccrC typing
–Generally performed by multiplex or simple PCR
methods and/or sequencing
November 2011 EURL course 31 DTU Food, Technical University of Denmark
SCCmec
• Staphylococcal Chromossomal Cassette:
– Integrated in the chromossome
– Variable in length – Different types in MRSA – Some SCC cassettes without
mec – Some SCCmec cassettes are
composites of different cassettes
– At present- types I-XI
November 2011 EURL course 32 DTU Food, Technical University of Denmark
Main concerns regarding MRSA in animal sector
• Studies indicate a large prevalence of MRSA in healthy animals in several countries – widespread in Europe and other parts of the world
• LA-MRSA found adapted to animal hosts • Finding in food products, even though considered low risk for
consumption and handling • Environmental persistence and colonization • Selection and co-selection factors not completely known
(Antimicrobials, metals, desinfectants?) • Increasing diversity of strains backgrounds and diversity of
SCCmecs • New mecA homologue in S. aureus from cattle UK
Large reservoir for Community acquired (CA)
MRSA infections even in Countries with low prevalence of MRSA
November 2011 EURL course 34 DTU Food, Technical University of Denmark
Reference laboratory activities on MRSA by EURL-AR
• Verification of strains from other European countries
• Provide courses including detection and typing of MRSA for
European labs
• Provide counselling to countries involved in MRSA studies
required by EC
• Proficiency testing
– MRSA EQAS ring trial on swab samples
– EQAS Staph- compulsory MRSA detection
• Advisory tasks
• Follow up on activities depending on EC decisions