A Guideline for the Molecular Typing of Canadian Epidemic Methicillin-Resistant Staphylococcus aureus Using spa Typing.

Transcription

1 A Guideline for the Molecular Typing of Canadian Epidemic Methicillin-Resistant Staphylococcus aureus Using spa Typing. George R. Golding National Microbiology Laboratory Winnipeg, Manitoba AMMI-CACMID Annual Meeting 2008

2 Characterization of MRSA Using PFGE Gold standard for the molecular typing of MRSA isolates.

3 Dic e ( O p t :. 0 0% ) ( To l. 0 % -. 0% ) ( H> 0. 0% S >0. 0 % ) [ 0. 0 % % ] PF G E - Sm ai % 80% 00% CMRSA6 CMRSA3 CMRSA7 PFGE Dendrogram of Epidemic CMRSA Isolates National PFGE database of over 4,000 MRSA isolates. CMRSA2 0 epidemic PFGE fingerprint types identified in Canada (CMRSA-0). CMRSA0 CMRSA5 CMRSA9 CMRSA-6, 8, and 9 are typically hospitalassociated. CMRSA CMRSA8 CMRSA4 CMRSA7 and 0 are typically communityassociated.

4 Disadvantages of PFGE Despite the excellent discriminatory power of PFGE, there are several disadvantages of using this technique, such as: a) long wait times to obtain final results. b) a high degree of labour intensity. c) lack of standardization between labs. d) inability to easily develop an MRSA typing database due to the subjective interpretation of results.

5 spa Typing spa-typing is a DNA sequencing technique that examines the polymorphic X, or short sequence repeat (SSR), region of the protein A gene (spa). 3F 54R C X X 2 X n

6 Changes in the SSR regions can arise due to A) deletions, B) duplications and C) point mutations 3F 54R C X X 2 X n t00w 3F 54R A) C X X n t00x 3F 54R B) C X X 2 X 2 X n t00y 3F 54R C) C X X 3 X n t00z This results in a diverse collection of spa-types where each spa-type consists of a specific combination of SSRs

7 Purpose The purpose of this study was to determine if spa typing could be used as an alternative to PFGE for the typing of Canadian epidemic MRSA (CMRSA) isolates.

8 MRSA Isolate Selection 488 Canadian epidemic MRSA isolates were selected from a 3 year collection (995 to 2007) of over 4,000 isolates CMRSA CMRSA2 CMRSA3 CMRSA4 CMRSA5 CMRSA6 CMRSA7 CMRSA8 CMRSA9 CMRSA0 Number of Tested Isolates

9 Guideline Development ) Correlation of spa with a specific PFGE fingerprint type? 2) Correlation of spa clonal clusters with PFGE epidemic CMRSA types? 3) Correlation of spa types with PFGE epidemic types?

10 ) Correlation of spa with a specific PFGE fingerprint type? Number of Tested PFGE Finger Print Types Number of spa Types CMRSA CMRSA CMRSA3 5 3 CMRSA CMRSA CMRSA CMRSA CMRSA CMRSA CMRSA0 44 8

11 ) Correlation of spa with a specific PFGE fingerprint type? Dice (Opt:.00%) (Tol.0%-.0%) (H>0.0% S>0.0%) [0.0%-00.0%] spa PFGE typing - SmaI of multiple isolates (range 2-9) from the same PFGE fingerprint types were determined. Epidemic TypeSpaType RepeatSuccession CMRSA2 CMRSA2 CMRSA2 CMRSA2 CMRSA2 t242 t002 t067 t002 t002 r26r23r7r3r7r20r7r2r7r6 r26r23r7r34r7r20r7r2r7r6 r26r23r7r34r7r20r7r2r7 r26r23r7r34r7r20r7r2r7r6 r26r23r7r34r7r20r7r2r7r6 In total, 89/256 (35%) tested indistinguishable PFGE patterns contained anywhere from 2-5 different spa types. As a result, spa typing could not reliably be used as an indicator of a specific PFGE fingerprint type.

12 2) Correlation of spa clonal clusters with PFGE epidemic CMRSA types? CMRSA5, 9, 0 CC6 2 2 CMRSA CC Number of Isolates > CMRSA3, 4, 6 CC5 CMRSA2 CC2 2 6 CMRSA8 CC4 4 CMRSA7 CC

13 3) Correlation of spa types with PFGE epidemic CMRSA types? spa Type Number of PFGE epidemic Types PFGE Epidemic Type(s) Number of Isolates % t002 N=497 CMRSA t003 N=4 CMRSA t004 N=24 CMRSA t005 N= CMRSA8 00 t007 N=3 CMRSA t008 CMRSA N=245 3 CMRSA CMRSA t00 N=4 CMRSA t02 N=38 CMRSA t04 N=4 CMRSA t05 N=2 CMRSA 2 00 t08 N=35 CMRSA t09 N=5 CMRSA t020 N= CMRSA8 00 t02 N=7 CMRSA t022 N=8 CMRSA The majority of spa types (0/4) were only associated with a single PFGE epidemic type. Basis for the preliminary guideline.

14 Guideline assignment of spa types belonging to multiple epidemic types e.g. spa type t008: identified within CMRSA5, CMRSA9, and CMRSA0 PFGE epidemic fingerprint types. To date PVL not identified in CMRSA5 and CMRSA9 strains. PVL-ve t008 PFGE PVL+ve CMRSA5 CMRSA9 CMRSA0 CMRSA0

15 Validation of the Guideline The first 300 MRSA isolates received in 2007 through CNISP was used to validate the spa typing guideline. No. Isolates t002 t003 t004 t008 t02 t037 t088 t28 others (<=2) spa Type

16 Validation of the guideline spa types using the guideline and PFGE banding patterns were independently assigned CMRSA epidemic types and then compared. Assigned as a CMRSA Epidemic strain Assigned as a Non- Epidemic strain spa PFGE Examination of the PFGE dendrograms revealed that of the 7 non-epidemic PFGE assignments actually clustered within an epidemic type, which was the same epidemic type predicted using spa typing. International comparison of spa types also revealed that two of the six non-epidemic assigned isolates were actually pandemic CA-MRSA strains (ST59 and ST80).

17 Guideline Summary Assignment of Canadian MRSA epidemic types based solely on the spa typing guideline was highly successful and therefore we recommend its use for surveillance purposes. Replacement of PFGE with spa typing for outbreak investigations was not investigated in this study and therefore can not be recommended. This spa typing guideline will be continually updated and readily available upon request.

18 Advantages of spa typing in comparison to PFGE ) Using current definitions; PFGE alone potentially misclassified 4.3% of MRSA isolates as sporadic. 2) Speed and ease of use. 3) DNA sequences provide unambiguous data interpretation. 5) The ability to easily develop and compare MRSA typing databases on a national and global scale.

19 International Comparison spa type t002 t004 t008 Other associated MRSA Clones CC5, Rhine Hesse MRSA (prototype), EMRSA-3, New York clone, Japan clone, Pediatric, USA00 ORSA II, USA800 ORSA IV, ST 5 ORSA I CC45, Berlin MRSA (prototype), USA600 ORSA II, USA600 ORSA IV CC8, Northern German MRSA (subclone), USA300 ORSA IV (cmrsa in the US), Archaic/Iberian, ST250 ORSA I Canadian Epidemic Type CMRSA2 CMRSA CMRSA9, CMRSA0 t08 CC30, prototype of ST-36, EMRSA- 6, USA200 ORSA II CMRSA4 t032 t037 t064 Barnim MRSA (prototype & subclone), EMRSA-5, prototype of ST-22, CC22 CC8/239, Vienna MRSA, Brazilian/Hungarian, ST239 ORSA III, ST240 ORSA III, EMRSA-, -4, -7, -9, - CC8, Archaic/Iberian, USA500 ORSA IV, USA500 ORSA II, ST8 ORSA I, ST8 ORSA IV, ST8 ORSA III CMRSA8 CMRSA3 CMRSA5 t28 ST (related to USA400/MW2) CMRSA7

20 Acknowledgements Dr. Michael Mulvey (NML, Winnipeg) Jennifer Campbell and Dave Spreitzer (NML, Winnipeg) Joe Vehyl and Kathy Surynicz (U of Manitoba) Dr. Andrew Simor (Sunnybrook, Toronto) DNA Core Facility (NML, Winnipeg) Canadian Nosocomial Infection Surveillance Program (CNISP)