Identification of a Novel Variant of Staphylococcal Cassette ...

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Jun 13, 2008 - highly homologous to that of type I.2 SCCmec of strain PL72 (previously ... two novel SCCmec elements, a type II SCCmec variant and a.
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, June 2009, p. 2616–2619 0066-4804/09/$08.00⫹0 doi:10.1128/AAC.00772-08 Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Vol. 53, No. 6

Identification of a Novel Variant of Staphylococcal Cassette Chromosome mec, Type II.5, and Its Truncated Form by Insertion of Putative Conjugative Transposon Tn6012䌤† Xiao Han,1 Teruyo Ito,1,2* Fumihiko Takeuchi,1 Xiao Xue Ma,2 Michihiko Takasu,2 Yoshio Uehara,3 Duarte C. Oliveira,4,5 Hermínia de Lencastre,5,6 and Keiichi Hiramatsu1,2 Department of Infection Control Science, Graduate School of Medicine,1 and Department of Bacteriology, School of Medicine,2 Juntendo University, 2-1-1 Hongo, Bunkyo-ku, Tokyo, Japan; General Medicine, Kochi Medical School, Kochi, Japan3; Centro de Recursos Microbiolo ´gicos (CREM), Faculdade de Cieˆncias e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal4; Instituto de Tecnologia Química e Biolo ´gica (ITQB), Universidade Nova de Lisboa, 2780-156 Oeiras, Portugal5; and The Rockefeller University, New York, New York6 Received 13 June 2008/Returned for modification 19 July 2008/Accepted 29 March 2009

We identified two novel staphylococcal cassette chromosome mec (SCCmec) elements in sequence type 8 methicillin-resistant Staphylococcus aureus strains isolated in Japan: type II.5 SCCmec, whose J1 region was highly homologous to that of type I.2 SCCmec of strain PL72 (previously isolated in Poland), and its J1 region variant caused by the deletion/insertion of putative conjugative transposon Tn6012, identified in four S. aureus genomes.

long-range PCR with the primer pairs indicated in Fig. 1 and Table 1 and determined the nucleotide sequences of the regions upstream of their mecA genes. Since a preliminary BLAST search suggested that the J1 region of the JCSC6833 SCCmec was homologous to that of the SCCmec element of strain PL72, isolated in 1991 at the University Children’s Hospital in Warsaw, Poland (8), and reported to be a type I SCCmec variant (type I.2) (9), we have also resequenced parts of the SCCmec of strain PL72 for comparative purposes. Nucleotide sequences were determined as described previously (5). The overall structures of the SCCmec elements of JCSC6826, JCSC6833, and PL72 are illustrated in Fig. 1. These elements were integrated downstream of orfX, at exactly the same position as those of other known SCCmec elements, and carried characteristic sequences, direct repeats and inverted repeats, at both ends. The sizes of the three elements, calculated from the sizes of amplified DNA fragments and nucleotide sequences, are 28, 39, and 25 kb, respectively. The regions from orfX to mecA of the SCCmec elements of JCSC6833 and JCSC6826 were the same as that of the SCCmec of strain N315. They carried plasmid pUB110, which was flanked by integrated copies of IS431. The JCSC6833 SCCmec carried mecA, mecR1, and mecI genes, which are essential components of the class A mec gene complex, and type 2 ccr. In addition, the region from mecR1 to ccr was identical to that of type II.1 SCCmec. However, the J1 region of the JCSC6833 SCCmec, which included four open reading frames (ORFs) with unknown functions, was identical to that of the SCCmec of PL72, which was reclassified as a variant of type I SCCmec, with degrees of nucleotide identity over 99.8% (see Tables S1 and S2 in the supplemental material) (9). To date, four distinct J1 regions in type II SCCmec elements have been identified: those of

Recent advances in the field of genetics of methicillin-resistant Staphylococcus aureus (MRSA) enable us to distinguish MRSA clones by examining the genotype of the strain, e.g., by multilocus sequence typing or pulsed-field gel electrophoresis, and the type of staphylococcal cassette chromosome mec (SCCmec) integrated into the chromosome. This study was undertaken to determine the structures of two novel SCCmec elements, a type II SCCmec variant and a nontypeable SCCmec. Eight MRSA isolates, isolated in June to August 2004 from four patients at hospitals in Kochi prefecture in Japan, were investigated in this study. These isolates were susceptible to clindamycin and cefozopran, in contrast to the dominant resistant strains; belonged to the same sequence-based genotype group, Spa type t008, coagulase type 3, and sequence type 8; and showed the same pulsed-field gel electrophoresis banding patterns for SmaI digests, which were distinct from those of the dominant strains. Seven isolates from three patients (represented by JCSC6833) carried a type II SCCmec whose J1 region (the region from ccr to the right-flanking chromosome) could not be classified into known subtypes, and one isolate (JCSC6826) from one patient carried an SCCmec in which known types of ccr genes could not be identified. We have amplified DNA fragments covering entire regions of SCCmec elements of JCSC6833 and JCSC6826 by

* Corresponding author. Mailing address: Department of Bacteriology, Juntendo University, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan. Phone: 81-3-5802-1041. Fax: 81-3-5684-7830. E-mail: teruybac @juntendo.ac.jp. † Supplemental material for this article may be found at http://aac .asm.org/. 䌤 Published ahead of print on 13 April 2009. 2616

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FIG. 1. Structures of two novel genetic elements in comparison with those of type II.1 and type I.2 SCCmec elements. Structures of SCCmec elements were illustrated based on the nucleotide sequences determined in this study and the sequence of the SCCmec element of strain N315 (DDBJ/EMBL/GenBank accession no. D86934). The locations of SCC integration site sequences are shown by red arrowheads. Other arrowheads indicate the locations of primers; black arrowheads represent primers for long-range PCR amplification of DNA fragments in each SCCmec element, and green arrowheads represent primers for nested PCR used for the identification of the closed circular form of Tn6012. Yellow arrows indicate transposases, red arrows indicate ORFs in the type 2 ccr gene complex, blue arrows indicate ORFs in the type 1 ccr gene complex, orange arrows indicate potassium-dependent ATPase operons used as representative makers of subtype 1 of type II SCCmec, purple arrows indicate ORFs in the J1 region common to type I.2 and II.5 SCCmec elements, gray arrows indicate mec regulator gene mecR1 or mecI, and green arrows indicate ORFs homologous to ORFs related to conjugative plasmids or conjugative transposons.

the SCCmec of N315 (subtype 1), the SCCmec of JCSC3063 (subtype 2), the SCCmec of strain AR13.1/3330.2 (subtype 3), and the SCCmec of strain RN7170 (subtype 4) (4–6, 11). Accordingly, we have designated the element of JCSC6833 type II.5.1.1 SCCmec, as an element carrying class A mec and type 2 ccr, a fifthly identified J1 region, and J2 and J3 regions in common with those of the SCCmec element of N315. This is the fourth case that has shown how the same J1 region may be shared by distinct SCCmec types, after the cases of type II.3 SCCmec and type IV.2 SCCmec (11), type II.2 SCCmec and type IV.7 SCCmec (1), and type I.2 SCCmec and type IV.8 SCCmec (1). These data suggested that a region demarcated by two direct repeats which did not carry ccr might have acquired ccr independently to form an SCC and that, by the acquisition of a distinct mec gene complex, such SCCmec elements would be generated. The JCSC6826 SCCmec element carried mecA, a mecR1 deletion, and the right end of the J1 region in common with that of the JCSC6833 SCCmec or PL72 SCCmec. The region from the 3⬘ end of the mecR1 gene to the right end of SCCmec (which includes the J2 region, the ccr locus, and the J1 region) was replaced by a DNA segment highly homologous (97.8 to 98.8%) to segments found in the genomes of

four S. aureus strains, COL, MRSA252, USA300-FPR3757, and USA300-TCH1516. Therefore, we regard the SCCmec of JCSC6826 as a pseudo type II.5 SCCmec. The homologous segments in the four S. aureus genomes were found at different locations, and all shared characteristic nucleotide motifs at both extremities, including inverted repeats of 2 bp (GG and CC) and 3-bp direct repeats (TAT for MRSA252 and AAT for the other three strains) that might be generated by target duplications. The corresponding segment present in the SCCmec of JCSC6826 shared the 2-bp inverted repeats at both extremities, one (GG) located at 875 bp from the start codon of the mecR1 gene and the other (CC) located at the extremity of the J1 region, although the 3-bp direct repeats were not observed. The sizes of these segments ranged from 13,339 to 13,355 bp. The segments encoded putative proteins related to transposition or DNA transfer (Table 2), including integrase, a truncated transposase, the product of traG, which encodes a coupling protein in conjugative plasmids pSK41 and pGO, an FtsK/ SpoIIIE family protein, and a relaxase that forms singlestranded DNA (T-DNA), and also carried six ORFs that were homologous to ORFs of conjugative transposons

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HAN ET AL. TABLE 1. List of primers used in this study Region amplified or purpose of primer

Regions of SCCmec carried by JCSC6833 orfX to pUB110 pUB110 to mecA mecA to Tn554 Tn554 to ccr ccr to chromosome Regions of SCCmec carried by PL72 orfX to mecA mecA to ccr ccr to chromosome Regions of pseudo SCCmec carried by JCSC6826 orfX to pUB110 pUB110 to mecA mecR1 to chromosome Identification of Tn6012 Set 1 Set 2 Set 3 Set 4 Set 5 Identification of closed circular formation of Tn6012 First round Second round

Name of primer

Nucleotide sequence (5⬘ to 3⬘)

cR1 ant2 ant1 mA1 mA2 ermA1 ermA2 pSJ21-4 pSJ21-3 cL4

AAGAATTGAACCAACGCATGA CTGCTAAATCGGTAGAAGC CAGACCAATCAACATGGCACC TGCTATCCACCCTCAAACAGG AACGTTGTAACCACCCCAAGA TGAAACAATTTGTAACTATTGA TGGGTAAACCGTGAATATCGTGT ATTGACCCTAAAACTAACACTGA TATCTCTTAAGGCGTTGACAACAT CAGTCGCATCAAATGTCTCTAATG

cR1 mA1 mA2 KT1 ␤2 cL1

AAGAATTGAACCAACGCATGA TGCTATCCACCCTCAAACAGG AACGTTGTAACCACCCCAAGA CGTTATGCAGTCATTACAAC ATTGCCTTGATAATAGCCTTCT ATTTAATGTCCACCATTTAACA

cR1 ant2 ant1 mA1 mCR3 cL1

AAGAATTGAACCAACGCATGA CTGCTAAATCGGTAGAAGC CAGACCAATCAACATGGCACC TGCTATCCACCCTCAAACAGG GTCTCCACGTTAATTCCATT ATTTAATGTCCACCATTTAACA

cT1 cT2 cT3 cT4 cT5 cT6 cT7 cT8 cT9 cT10

ATATCATTGCTATCGTCTTG ATGTTTACTCTTGTAACAG CATTGACTCTTGTTGTTCTC ATGAATCATATACGTAATCA TCATCACCAATAACGCGCTT GGTTCGAGGTTTCAAGATAA GGATCCCAGTACAACATTTG ACCGTGCGAAAATAAATCAT TCATGATATTCTACTTCTTCATCA TCCCTGATAGTCAATTGAAT

cT11 cT12 cT13 cT14

CCAATGTAGTTTGTAATGAG GTCTAATTCCACTTTATTAG GCCTCGAATGATATTTGAC GGTGTAAACTCAGTCATAATTA

Tn916 and Tn5801 (2, 7). The formation of a closed circular intermediate with head-to-tail ligation of both termini, which might be generated upon excision of the segment, was confirmed by a nested PCR experiment (Table 1) and subsequent sequencing of the chromosomal DNA nucleotides of three strains, namely, JCSC6826, COL, and MRSA252. Consequently, we designated the JCSC6826 SCCmec segment putative conjugative transposon Tn6012. Tn6012 was distinct from Tn916 or Tn5802 as follows: the int genes of the transposons were not homologous, Tn916 does not duplicate the target site upon integration (10), an excisionase gene was absent in Tn6012, traG was present in Tn6012, and the orders of ORFs differed. Tn6012 was identified in four S. aureus strains with sequenced genomes but was not found in the genomes of other

Size (kb) of amplified DNA fragment

7 4.5 9 11.7 8

10 9 9

7 4.5 15

0.6 0.9 0.4 0.6 1

1.3 0.4

species, suggesting that it may be specific for staphylococci. When we investigated the dissemination of Tn6012 by PCR with a pair of primers identifying the ORF ftsK/spoIIIE, fs1 (5⬘-TCATCACCAATAACGCGCTT-3⬘) and fs2 (5⬘-GGTTC GAGGTTTCAAGATAA-3⬘), 2 of 47 S. aureus strains gave positive results, suggesting that Tn6012 was rather widely disseminated in S. aureus strains. The absence of specific genes related to antibiotic resistance or pathogenicity seemed to be the reason why it was not noticed as a genomic island until two USA300 clones were compared (3). Nucleotide sequence accession numbers. The nucleotide sequences of the three SCCmec elements determined in this study were deposited in the DDBJ and EMBL and GenBank databases under accession no. AB435014 (JCSC6826 SCCmec), AB435013 (JCSC6833 SCCmec), and AB433542 (PL72 SCCmec).

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TABLE 2. ORFs located upstream of mecA in SCCmec of JCSC6826a ORF

Gene size (bp)

Location

Length of product (aa)

Gene designation mecR1 tnp

KC01* KC02*

54–927 1148–2168 (complement)

874 1,021

KC03 KC04 KC05

2277–2618 (complement) 2674–3264 (complement) 3371–4317 (complement)

342 591 1,047

113 196 348

KC06

4307–6154 (complement)

1,848

615

KC07

6159–7517 (complement)

1,359

452

KC08

7571–10066 (complement)

2,496

831

KC09

10101–10484 (complement)

384

127

KC10

10496–10756 (complement)

261

86

KC11

10761–11816 (complement)

1,056

351

KC12

11877–12968 (complement)

1,092

363

KC13 KC14 KC15 KC16

13143–13445 (complement) 13459–13779 (complement) 13930–14214 (complement) 14361–14858

303 321 285 498

100 106 94 165

traG

ftsK/spoIIIE

Name and/or description of product (Truncated) MecR1 (Truncated) integrase, catalytic region Hypothetical protein Hypothetical protein Putative TraG membrane protein Hypothetical membrane protein FtsK/SpoIIIE family protein Hypothetical membrane protein Hypothetical membrane protein Hypothetical membrane protein Hypothetical membrane protein Replication initiation factor family protein Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein

Corresponding ORF in conjugative transposon Tn916 (size in bp)

% Identityb

Corresponding ORF in conjugative transposon Tn5801 (size in bp)

% Identityb

orf15 (2,265)

46.50

SAV401 (2,028)

48.00

orf21 (1,386)

51.20

SAV409 (1,404)

51.60

orf16 (2,448)

47.20

SAV402 (2,454)

48.10

orf13 (933)

45.80

SAV399 (963)

49.20

orf20 (990)

49.80

SAV408 (1,185)

50.90

orf22 (387)

50.30

SAV411 (378)

48.20

a Data for coding sequences are given. Nucleotide positions are based upon the JCSC6826 nucleotide sequence deposited under DDBJ and EMBL and GenBank database accession no. AB435014, and they correspond to the 5⬘ to 3⬘ direction. *, pseudogenes (sizes of reconstituted ORFs are indicated); aa, amino acids. b Identity of the nucleotide sequence to that of each ORF.

This work was supported by a grant-in-aid for 21st Century COE research and a grant-in-aid for scientific research, no. C19590456, from the Ministry of Education, Science, Sports, Culture and Technology of Japan.

6.

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