Mosaic Staphylococcal Cassette Chromosome mec Containing Two Recombinase Loci and a New mec Complex, B2

Abstract

A novel staphylococcal cassette chromosome (SCC) mec from a clinical methicillin-resistant Staphylococcus aureus isolate (ST100/CC5) had a mosaic structure, composed of SCC DNA from several different backgrounds. It harbored two complete ccr loci and a new variant of mec complex B, with ΔmecR1 interrupted by the aminoglycoside resistance transposon Tn4001.

Methicillin resistance in Staphylococcus aureus (MRSA) is facilitated by the acquisition of the staphylococcal cassette chromosome mec (SCCmec), which integrates site specifically into the staphylococcal genome and carries mecA, encoding the alternative penicillin-binding protein PBP2a, a β-lactam-insensitive transpeptidase (6, 11, 13, 22, 26). The precise excision and site- as well as orientation-specific integration of this element depend on the action of cassette chromosome recombinase genes (ccr's) located within the element (13).

Five main types of SCCmec have been described so far, each differing in size and composition and characterized according to its type of ccr locus and mec complex (3, 7, 25). mec complexes differ in the extents of insertion sequence (IS)-mediated deletions in the mecA regulatory genes mecR1 and mecI and the presence and location of insertion sequence IS431, IS1182, or IS1272 (23). Apart from the ccr and mec complexes, and some common mobile resistance elements, SCCmec subtypes harbor variable J (junkyard) regions containing truncated and nonessential genes and genes of unknown functions (8). In addition to the major types, a number of new SCC elements, including non-mecA-carrying cassettes, have recently been discovered (4, 5, 9, 14, 15, 17, 19).

An epidemiological study of methicillin-resistant staphylococci from Zurich in 2003 identified several strains which contained multiple ccr loci (21). Here, we describe the SCCmec of one of these isolates, MRSA_ZH47.

MRSA_ZH47 is of multilocus sequence type 100 and belongs to clonal complex 5, a genotype previously identified in Argentina (1, 24). In addition to its β-lactam resistance, it was resistant to aminoglycosides and carried a blaZ-encoded penicillinase. Its SCCmec type could not be determined by standard multiplex PCR (20), and additional ccr typing indicated that it contained both ccr2 and ccrC loci (21).

Southern hybridization of SmaI-digested chromosomal DNA, separated by pulsed-field gel electrophoresis, showed that mecA- and ccr2-hybridizing sequences were colocated on a separate SmaI fragment from the ccrC-hybridizing sequence (data not shown).

Transient overexpression of ccrAB2, facilitating the precise excision of all major SCCmec types (10, 12, 13, 18), was used to cure MRSA_ZH47. Southern hybridization showed that the resulting oxacillin-susceptible clone MRSA_ZH47c had lost mecA and both ccr loci, indicating that all three were present on a single excisable SCC element containing an internal SmaI restriction site (data not shown). The susceptibility profile of MRSA_ZH47c showed that aminoglycoside resistance had also been lost.

A cosmid library of MRSA_ZH47 DNA, consisting of over 600 clones with estimated inserts of about 45 kb, was constructed using a SuperCos1 cosmid vector kit (Stratagene, La Jolla, CA). Screening of the library by colony blot analysis using ccr2- or ccrC-specific probes identified 11 clones that hybridized to ccr2, 7 that hybridized to ccrC, and 2 that hybridized to both probes. Cosmids were end sequenced and the sequences compared to the genome sequence of S. aureus Mu50, revealing that the two cosmids hybridizing with both probes each contained one end of the SCCmec element and together completely covered it (data not shown).

Primers specific for known orfX, ccrC2, IS431, and mecA nucleotide sequences were used to synthesize long-range PCR products that were subcloned into either pUC19 or pBluescript SK(+). Inserts were end sequenced and the obtained sequences assembled. The double-stranded nucleotide sequence of the 33.7-kb element was completed by primer walking.

This SCCmec proved to be unique, containing elements and properties not previously described. GeneMark.hmm (16) and BLASTX (2) identified 33 open reading frames (ORFs), all of which were identical or highly similar in sequence to previously annotated staphylococcal genes (Table 1 and Fig. 1).

TABLE 1.

ORFs encoded on SCCmec_ZH47

Coding sequence no.	Namea	Position (bp)	Identity (%)b	Homolog(s)c	Informationd
	DR-L	754-771			Integration site sequence of SCCmec, A→G substitution at position 16 of left direct repeat
	IR-L	757-764			Integration site sequence of SCCmec; left inverted repeat
1	ZH02	1017-1322	98	CZ078 (85/2082)	Hypothetical protein, predicted restriction endonuclease domain (COG 3183)
2	ZH03	1887-2384	100	CZ077 (85/2082)	Conserved hypothetical protein (COG 3680)
3	ZH04	2468-3967	100	CZ076 (85/2082)	Hypothetical protein
4	ZH05	4193-5293	100	CZ075 (85/2082)	Hypothetical protein; DNA polymerase A family domain (Pfam 00476.12)
5	ZH06	5286-5657	94	CZ074 (85/2082)	Hypothetical protein (DUF 1092)
6	ZH07	5654-7273	100	3 half: CG008 (85/3907)	Hypothetical protein; POX_D5 domain associated with viral DNA replication (Pfam 03288.11)
100	5 half: unnamed ORF (TSGH17)
7	ccrC	7498-9174	94	ccrC2 (TSGH17)	Cassette chromosome recombinase C
			89	ccrC3 (85/2082)
8	ZH09	9280-9618	98	SSP0034 (ATCC 15305)	Hypothetical protein
9	ZH10	9714-10025	90	SSP0032 (ATCC 15305)	Hypothetical protein, contains SmaI restriction site
10	ZH11	10041-10547	90	CZ068 (85/2082)	Conserved in gram-positive and -negative bacteria but of unknown function (COG 4333)
11	IS431	10696-11370	99	IS431 (SCCmec types I-IV)	Insertion sequence IS431
12	ZH13	11628-11795	100	ORF CN041 (N315) and in SCCmec types I-V	Putative HMG-coenzyme A-synthase (cholesterol biosynthesis)
13	ugpQ	12712-13455	100	ugpQ (all SCCmec types)	Glycerophosphoryl diester phosphodiesterase
14	ZH15	13552-13980	100	SA0037 (N315) and in SCCmec types I-V	Hypothetical protein, MaoC-like domain (Pfam 01575.11)
15	mecA	14026-16032	100	WIS (SCCmec type V) and TSGH17 (SCCmec type VT)	Penicillin-binding protein PBP2a
16	ΔmecR1′	16132-16958	100	ΔmecR1 from mec complex B	First 826 bp of ΔmecR1, truncated signal transducer MecR1 from mec complex B
	DRTn4001	16951-16958			Repeated region generated by transposon integration
	IRTn4001	16959-17059			Repeated region generated by transposon integration
17	IS256L	17060-18232	100	Tn4001	IS of Tn4001
18	aac	18235-18681	100	Tn4001	Putative N-acyltransferase, GNAT family
19	aac(2′)-aph(6")	18682-20121	100	Tn4001	Aminoglycoside-(2′)-acetyltransferase-aminoglycoside-(6")-phosphotransferase
20	IS256R	20251-21423	100	Tn4001	IS of Tn4001
	IRTn4001	21424-21524			Repeated region generated by transposon integration
	DRTn4001	21525-21532			Repeated region generated by transposon integration
21	ΔmecR1"	21533-21692	100	ΔmecR1 from mec complex B	Remainder of ΔmecR1, interrupted by Tn4001 insertion
22	ZH22	21595-21924	100	ΔhsdR (MW2)	Truncated hypothetical protein, similar to type I restriction endonuclease
23	ZH23	21915-23438	100	IS1272 transposase from mec complex B	aa 1-102: transposase and inactivated derivatives (COG 3666)
					aa 185-459: transposase DDE domain
24	ZH24	23574-24083	100	Various type IV SCCmecs	Hypothetical protein
25	ZH25	24095-24406	100	Various type IV SCCmecs	Hypothetical protein
26	ZH26	24493-24843	100	Various type IV SCCmecs	Hypothetical protein
27	ccr2B	25365-26993	100	ccrB SCCmec type IVd (JCSC4469)	Cassette chromosome recombinase B
28	ccr2A	27015-28364	100	ccrA SCCmec type IVe (AR43)	Cassette chromosome recombinase A
29	ZH29	28598-30391	100	R004 (MR108)	aa 1-382: superfamily II helicase and inactivated derivatives (COG 5519)
30	ZH30	30391-30687	100	Different type IV SCCmecs	Hypothetical protein
31	ZH31	30880-31926	100	Different type IV SCCmecs (ATCC 12228)	Hypothetical protein
32	ZH32	32381-33535	100	CR008 (MR108)	aa 1-122: abi alpha protein, predicted transcriptional regulator (COG 2865)
33	ZH33	33565-34365	100	Different type IV SCCmecs and SE0042 (ATCC 12228)	Hypothetical protein, abortive phage resistance protein
	IR-R	34423-34430			Integration site sequence of SCCmec; right inverted repeat
	DR-R	34432-34449			Integration site sequence of SCCmec; right direct repeat

^aORFs of known functions are named accordingly; putative and hypothetical ORFs have been assigned ZH numbers.

^bAmino acid sequence identity.

^cThe name of the homologous gene is indicated, and the strain in which it was found is indicated in parentheses. If homologous ORFs were unnamed, the SCCmec type in which they were found is indicated.

^dInformation about the element, e.g., about the encoded protein. Positions and reference numbers of known protein domains are indicated. aa, amino acids; HMG, 3-hydroxy-3-methylglutaryl; COG, cluster of orthologous groups of proteins.

FIG. 1.

Genetic map of the mosaic SCCmec element from MRSA_ZH47. Open reading frames are symbolized by arrows, and the bars below them indicate homologies to previously described SCC elements. Integration site sequences for SCCmec (*), direct repeats of chromosomal junctions (◂), and inverted repeats (▹) are indicated. J_L denotes the junction proximal and J_R the junction distal to the origin of replication.

The orfX insertion site and the characteristic terminal inverted and direct repeats, generated upon insertion, were almost identical to those of other, previously described SCCmec elements (12). However, the left-end (proximal) direct repeat sequence contained a nucleotide transition of an adenine to guanine (Table 1), which has not been found elsewhere and increased the identity between the junctional direct repeats. This mutation did not impede the excision of the element, as demonstrated by the precise curing of SCCmec_ZH47 from the chromosome of MRSA_ZH47. It could, however, possibly influence the stability or transfer frequency of the element.

SCCmec_ZH47 contained a new mec complex that we have named B2 because of its similarity to mec complex B. The new B2 complex differed in that the 987-bp ΔmecR1 fragment was interrupted by insertion of the aminoglycoside resistance transposon Tn4001 at base pair position 820 (Fig. 1), and while the mecA promoter region was identical to that of mec complex B, the mecA gene sequence was identical to that of SCCmec types V and V_T.

In addition to a ccrAB2 locus at the usual position downstream of mecA, the element possessed a ccrC locus between orfX and the dru element (Fig. 1). The ccrA2 sequence was identical to that of SCCmec type IVe, while the ccrB2 sequence was identical to that of SCCmec type IVd. Comparison of the ccrC sequence to published variants revealed high levels of similarity to the ccrC2 and ccrC3 sequences of SCCmec types V_T and III, respectively (Table 1).

The element as a whole appeared mosaic in structure. The presence of both a ccrAB2 locus and a variant ccrC locus and of regions with strong similarity to several different SCC elements, including the typical hospital-acquired MRSA type III SCCmec, the community-associated MRSA SCCmec types IV and V_T, and SCCmec from the non-S. aureus species Staphylococcus saprophyticus, suggests that SCCmec_ZH47 had been assembled via several recombination events (Fig. 1).

Most of the new SCC and SCCmec elements recently discovered, including the SCCmec_ZH47 described here, appear to have acquired regions from other SCC elements, suggesting that significant intra- and interspecies exchange and recombination of SCC DNA occurs.

Nucleotide sequence accession number.

The nucleotide sequence newly determined in this study was deposited in the EMBL database under accession number AM292304.