Transfer of the methicillin resistance genomic island among staphylococci by conjugation

Abstract

Methicillin resistance creates a major obstacle for treatment of Staphylococcus aureus infections. The resistance gene, mecA, is carried on a large (20 kb to > 60 kb) genomic island, staphylococcal cassette chromosome mec (SCCmec), that excises from and inserts site-specifically into the staphylococcal chromosome. However, although SCCmec has been designated a mobile genetic element, a mechanism for its transfer has not been defined. Here we demonstrate the capture and conjugative transfer of excised SCCmec. SCCmec was captured on pGO400, a mupirocin-resistant derivative of the pGO1/pSK41 staphylococcal conjugative plasmid lineage, and pGO400::SCCmec (pRM27) was transferred by filter-mating into both homologous and heterologous S. aureus recipients representing a range of clonal complexes as well as S. epidermidis. The DNA sequence of pRM27 showed that SCCmec had been transferred in its entirety and that its capture had occurred by recombination between IS257/431 elements present on all SCCmec types and pGO1/pSK41 conjugative plasmids. The captured SCCmec excised from the plasmid and inserted site-specifically into the chromosomal att site of both an isogenic S. aureus and a S. epidermidis recipient. These studies describe a means by which methicillin resistance can be environmentally disseminated and a novel mechanism, IS-mediated recombination, for the capture and conjugative transfer of genomic islands.

Fig. 1

(A) S. aureus N315, MR14, MR14EX. S. aureus strain N315 contains a 53 kb SCCmec type II element. The gene mecA encodes methicillin resistance; mecR1 and mecI encode mecA regulators; and ccrAB encode recombinases for SCCmec excision and integration. N315 was modified to construct MR14 for positive selection for excision. A promoterless tetracycline resistance gene, tetM, was inserted immediately downstream of attL, and Tn554 and 15.4 kb of DNA upstream of tetM were deleted. MR14EX is the excised tetracycline-resistant derivative of MR14 with tetM expression driven from the orfX promoter. (B) S. aureus MR14 spontaneous excision. Serial dilutions of an overnight MR14 culture were plated on TSA and TSA with tetracycline (TET). The frequency of spontaneous excision was the excised population (TET) divided by the total population (TSA). Bars indicate the standard deviation of three independent experiments. (C) S. aureus MR14 pWA46 inducible excision monitored by positive selection on tetracycline in the presence or absence of inducer (5μM cadmium chloride). Excision percentages were determined by plating overnight cultures on TSA and TSA with tetracycline and comparing growth on tetracycline to total growth without antibiotic selection. Bars indicate the standard deviation of three independent experiments. (D) Diagram of the PCR approach used to determine integrated, excised, and circular SCCmec. Primer sets 1 and 2 amplify attR and attL integrated junctions, respectively. Primer set 3 amplifies excised chromosomal junction (attB). Primer set 4 amplifies excised circular SCCmec junction (attS). (E) PCR analysis of strain MR14. Lane A, 1 kb molecular size marker; lanes 1 and 2, integrated attR and attL amplicons, respectively; lane 3, excised chromosomal junction (attB); lane 4, circular SCCmec junction (attS). (F) PCR analysis of strain MR14EX. Same lanes as in Fig. 1E. Lane 3 shows only the excised chromosomal junction (attB) is present. Lane 4 shows the circular SCCmec intermediate (attS), following excision, has been lost.

Fig. 2

Outline of filter matings for pRM27 transfer. First mating, original donor (MR14 pWA46 pRM27, apparent captured pGO400::SCCmec) with recipient 1 (N315EX, isogenic recipient with excised SCCmec). Resulting transconjugants 1 (N315EX pRM27) became the donor for a second mating with recipient 2 (RN4220 pCN36, heterologous recipient). The presence of different antibiotic resistance markers in the donor and recipient cells allowed for transconjugant selection. Tet, tetracycline; Chl, chloramphenicol; Mup, mupirocin; Kan, kanamycin; Nov, novobiocin; Rif, rifampin.

Fig. 3

(A) PCR analysis to characterize transconjugant N315EX pRM27. Lane A, 1 kb molecular size marker; lanes 1 and 2, integrated attR and attL amplicons, respectively; lane 3, excised chromosomal junction (attB); lane 4, circular SCCmec junction (attS); lane 5, mecA; lane 6, orf0042, specific to RN4220; lane 7, ermA, specific to N315; lane 8, mupA; lane B, 500 bp molecular size marker (B) PCR analysis to characterize transconjugant RN4220 pCN36 pRM27. The lanes are the same as in Fig. 3A.

Fig. 4

Southern hybridization analysis of NarI restriction-digested DNA separated by pulsed field gel electrophoresis. There is a single NarI cleavage site within pGO400 and no site within SCCmec. Lane A, MidRangeII PFG marker; lanes 1 N315EX pRM27, lanes 2, RN4220 pCN36 pRM27; lanes 3, RN4220 pGO400. The left panel was hybridized with a probe for mupA and the right panel for mecA.

Fig. 5

(A) pRM27 is a 64,127 bp plasmid consisting of the conjugative plasmid pGO400 (red) and captured, circularized SCCmec (blue). Loci of interest include the trs operon (purple) responsible for the conjugative transfer of the plasmid between strains and species; mupirocin resistance gene, mupA (green); IS elements (black), three from pGO400 (tnpC, tnpD, and tnpG) and two from SCCmec (tnpMec1 and tnpMec2). The insertion of SCCmec into tnpG and its duplication to form tnpMec2 are marked with red asterisks. The latter two flank pUB110 (grey) which also contains the gene for kanamycin resistance (aadD, light blue). Also shown are the mec operon (orange) and the ccr operon (pink) encoding CcrA and CcrB, the proteins responsible for site-specific recombination between attS (marked) and attB in the S. aureus genome. Accession number KT780705. (B) Chromosomal map of the conjugative plasmid pGO400 showing the 3 IS431/IS257 elements in black, tnpG, the IS element into which SCCmec inserted is marked with an asterisk, mupirocin resistance gene mupA in green and the genes of the transferase (trs) operon in maroon. Accession number KT780704.