การสอบสวนโรคต ดเช อในโรงพยาบาล

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1 การสอบสวนโรคต ดเช อในโรงพยาบาล MRSA Model in Rajavithi Hospital by Chanwit Tribuddharat, M.D., Ph.D. Department of Microbiology Faculty of Medicine Siriraj Hospital Mahidol University

2 Study Plan Infectious Disease experts from universities Microbiological experts from Thai NIH Volunteer hospital (Rajavithi Hospital) Infection Control personnel (Very very strong group of determined staff) Supporting budget from the Government (700,000 bahts) Accepted for publication in European Journal of Clinical Microbiology & Infectious Diseases on 30 April 2010

3 Study population and sample collection 619 in-patients 19 different wards During November 9 10, 2006 All participants have signed informed consent and agreed to be screened for the presence of MRSA from their stools nasal swabs The study has been approved from the ethical committee of the Rajavithi Hospital.

4 Microbiological Methods Nasal swabs, feces or rectal swabs were cultured on selective media (blood agar + 6 μg oxacillin + 4% NaCl) Feces or rectal swabs also were cultured on PEA (Phenylethyl Alcohol Agar) Confirmation of methicillin resistance was based on a 30 µg cefoxitin disk on Muller- Hinton agar and a 6 µg oxacillin disk

5 Phage typing 72 MRSA was performed by heat-shock technique (heating a culture at 55 C for 3 min immediately before phage typing) International phage typing set issued by the International Center, Colindale, UK was used

6 Determination of Hypervarible Region downstream of meca Gene primer HVR1: 5 -ACTATTCCCTCAGGCGTCC-3 (position ) Primer HVR2: 5 -GGAGTTAATCTACGTCTCATC-3 (position ) (Using GenBank accession number X52594)

7 Typing by Surface Protein A (spa) Gene spa forward primer: 5 -TGTAAAACGACGGCCAGTGCTAAA AAGCTAAACGATGC-3 spa reverse primer: 5 -CAGGAAACAGCTATGACCCCACCA AATACAGTTGTACC-3 Following by RsaI endonuclease restriction of the PCR product

8 Coagulase (coa) Gene Polymorphism Primers Coa2: 5 -CGAGACCAAGATTCAACAA G-3 Coa3: 5 -AAAGAAAACCACTCACATCA-3 Followed by AluI endonuclease restriction

9 Multi-locus sequence typing (MLST) Approximate bp PCR fragments of the seven housekeeping genes arc, aroe, glpf, gmk, pta, tpi, and yqil Allelic profiles were obtained from the MLST website

10 Results Total number of patients 619 Total identified carriers 57 MRSA in nose and stool 7 MRSA in nose only 45 MRSA in stool only 5

11 Confirmation 72 MRSA were isolated and confirmed by Chrom-agar coagulase production Latex Agglutination Test for identification of S. aureus (Pastorex Staph-plus : Bio-Rad) Latex Agglutination kit for the rapid detection of PBP2 (MRSA-Screen : DENKA SEIKEN CO.,LTD.) Oxacillin disk agar diffusion technique Oxacillin sreening agar test

12 Phage Typing Phage typing of 72 MRSA was performed by Heat-shock technique (heating a culture at 55 C for 3 min immediately before phage typing), using the international phage typing set issued by the International Center, Colindale,UK. The phage typing set consisted of Lytic group I : 29, 52, 52A, 79, 80 ; Lytic group II : 3A, 3C, 55, 71; Lytic group III: 6, 42E, 47, 53, 54, 75, 77, 83A, 84, 85 ; Lytic group V : 94, 96 Miscellaneous group : 81, 95. Susceptibility to phages was determined by standard routine test dilution (RTD) at 1,000 x RTD.

13 Phage Typing Of the 72 MRSA isolates, 50 (69.4%) nontypable, 19(26.4%) Lytic group III 3 (4.2%) Mixed group.

14 PCR typing methods SCCmec PCR Type I = 613 bp Type II = 398 bp Type III = 280 bp Spa gene PCR 504 bp Coa gene PCR 800 bp HVR PCR 291, 371,411, 451, 491, 531, 691, 771 bp

15 SCCmec PCR Primers Type I-F = 5 - gct tta aag agt gtc gtt aca gg -3 Type I-R = 5 - gtt ctc tca tag tat gac gtc c -3 Type II-F = 5 - cgt tga aga tga tga agc g -3 Type II-R = 5 - cga aat caa tgg tta atg gac c -3 Type III-F = 5 - cca tat tgt gta cga tgc g -3 Type III-R = 5 - cct tag ttg tcg taa cag atc g -3 PCR condition 95 C for 4 min 95 C for 1 min 65 C for 30 sec 10 cycles 72 C for 1 min 95 C for 1 min 55 C for 30 sec 25 cycles 72 C for 1 min 72 C for 5 min All of 72 strains revealed Type III SCCmec

16 Spa PCR Primers 5 -tgtaaaacgacggccagtgctaaaaagctaaacgatgc-3 5 -caggaaacagctatgaccccaccaaatacagttgtacc-3 PCR condition 95 C for 4 min 95 C for 40 sec 60 C for 30 sec 35 cycles 72 C for 1 min 72 C for 7 min All of 72 strains showed a 501 bp

17 Rsa I digested-spa PCR product All of 72 strains revealed A-pattern

18 Coa PCR Primers 5 -cgagaccaagattcaacaag-3 5 -aaagaaaaccactcacatca-3 PCR condition 95 C for 4 min 95 C for 40 sec 60 C for 30 sec 35 cycles 72 C for 1 min 72 C for 7 min All of 72 strains showed a 800 bp

19 Alu I digested-coa PCR product Pattern A = 1 strain Pattern B = 69 strains Pattern C = 1 strain Pattern D = 1 strain

20 HVR PCR Primers 5 -actattccctcaggcgtcc-3 5 -ggagttaatctacgtctcatc-3 PCR condition 95 C for 4 min 95 C for 1 min 55 C for 30 sec 35 cycles 72 C for 1 min 72 C for 7 min

21 HVR-PCR 3 DRU = 1 strain 5 DRU = 5 strains 6 DRU = 1 strain 7 DRU = 48 strains 8 DRU = 1 strain 9 DRU = 1 strain 13 DRU = 4 strains 15 DRU = 10 strains

22 Pulsed field gel electrophoresis (PFGE) CDC protocol. Extracted chromosomal DNA digested with the SmaI restriction enzyme. PFGE was performed with CHEF DRII and CHEF DRIII system (Bio-Rad laboratories), running condition was 6V/cm, with switching times of 5-40 seconds for 21 hours. PFGE was analyzed by Syngene Gene Directory Application Version

23 PFGE patterns 72 isolates

24 P33 Grouping by Combined PCR-based methods P36 P31(32) P11, 12, 30, 69, 44 P3, 53, 23, 25, 26, 27, 28, 41, 42, 46, P43 P52 P24, 45(72) P1, 2(51), 47(48, 49, 50), 4, 5, 6, 60, 7, 8, 9(10), 13, 14, 15(64), 20, 21, 61, 62,63,16, 17, 18, 19, 29, 34, 35, 37, 38, 39, 40, 54(55), 56(57), 58(59), 65, 66, 67(68), 70(71) P22

25 Floor plan of a 12-floor building Ward West wards East wards 10 th Floor, Ortho. (P34, ST1227), (P35, NoST ), (P37, (P46, NoST), (P45(P72), NoST) ST1227), (P38, NoST), (P39, ST239 ) 9 th Floor, ENT (P3, NoST), (P52(P53), ST239) 8 th Floor, Gen. Surg. 7 th Floor, Worker Healthcare/GYN 6 th Floor, Med. (P23, ST239), (P25, ST239), (P26, ST239), (P27, ST239), (P28, NoST), (P24, ST239) (P70(P71), NoST), (P43(P44), NoST) (P4, NoST), (P54(P55), ST239), (P56(P57), ST1228), (P58(59), ST239) 4 th Floor, Radio. (P40, ST239), (P41, NoST), (P42, NoST) 3 rd Floor, ICU. Surg. (P36, NoST) EMS Building West wards 3 rd Floor (P67(P68), NoST), (P30, ST239), (P69, NoST) Internal Medicine Building West wards (P7, ST239), (P8, NoST), (P9(P10), NoST), (P13, NoST), (P11, NoST), (P12, ST239) (P29, ST239), (P66, NoST) (P31(P32), ST239), (P33, ST239) East wards (P16, NoST), (P17, ST343) East wards (P5, NoST), (P6, ST239), (P60, NoST) 2 nd Floor 1 st Floor, ICU Med. (P14, NoST), (P15(P64), NoST), (P61, NoST), (P62, NoST), (P63, ST1227), (P20, ST239), (P21, NoST), (P22, ST241) (P1, NoST), (P47(P48, P49, P50), NoST), (P2(P51), ST239) (P18, NoST), (P19, NoST)

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27 ST239 and their complex ST239 8

28 Heteroduplex-PCR for ST239 MRSA ST 30-like specific primers SA0317Forward: 5 -TCGCACTCTCGTTGAACA-3 SA0317Reverse: 5 -AAATCCGCTTCGACAAACATT-3 ST 8-like specific primers SA2003Forward: 5 -CACTTTAAATACTGACGAAAAT-3 SA2003Reverse: 5 -TTGAAAATTGATCATTCAGCAA-3 Edward J Feil et al. J Clin Microbiol April; 46(4):

29 Heteroduplex PCR for ST239 Lineage ST-30 ST-8 ST-239 ST-241 ST-343 ST-1227 ST-1228 Like Like ST-30 Like = S. aureus Cowan I ST-8 Like = MRSA SCCmec IV ST239 = P24 ST241 = P22 ST343 = P17 ST1227 = P37 ST1228 = P56 ST8 F: 5 -CACTTTAAATACTGACGAAAAT-3 R: 5 -TTGAAAATTGATCATTC AGCAA-3 ST30 F: 5 -TCGCACTCTCGT TGAACA-3 R: 5 -AAATCCGCTTCGACAAACATT-3 The identification of the ST8 and ST30 recombinant chromosome. PCR of 484 bp = ST30-like chromosomal segment and 220 bp = ST8- like segment. T. Jariyasethpong, C. Tribuddharat, S. Dejsirilert, et al: Eur J Clin Microbiol Infect Dis (2010) 29:977 9

30 Conclusions MRSA is a major nosocomial pathogen At the prevalence rate of 9.2%. Risk factors for MRSA carriage were the same as other studies (long stay, Antibiotics, male, chronic diseases, etc.) Long term and large scale spread of MRSA subtypes has occurred in a single hospital. The predominant MRSA type in this hospital is of ST239 clonal cluster.