Antibiotics trigger initiation of SCCmec transfer by inducing SOS responses

Abstract

The rise of antimicrobial resistance limits therapeutic options for infections by methicillin-resistant staphylococci. The staphylococcal cassette chromosome mec (SCCmec) is a mobile genetic element as the only carrier of the methicillin-resistance determinants, the mecA or mecC gene. The use of antibiotics increases the spread of antibiotic resistance, but the mechanism by which antibiotics promote horizontal dissemination of SCCmec is largely unknown. In this study, we demonstrate that many antibiotics, including β-lactams, can induce the expression of ccrC1 and SCCmec excision from the bacterial chromosome. In particular, three widely used antibiotics targeting DNA replication and repair (sulfamethoxazole, ciprofloxacin and trimethoprim) induced higher levels of ccrC1 expression and higher rates of SCCmec excision even at low concentrations (1/8 × minimum inhibitory concentration). LexA was identified as a repressor of ccrC1 and ccrAB by binding to the promoter regions of ccrC1 and ccrAB. The activation of RecA after antibiotic induction alleviated the repression by LexA and increased the expression of ccrC1 or ccrAB, consequently increasing the excision frequency of the SCCmec for SCCmec transfer. These findings lead us to propose a mechanism by which antimicrobial agents can promote horizontal gene transfer of the mecA gene and facilitate the spread of methicillin resistance.

Figure 1.

Antibiotics and Ultraviolet (UV) exposure promote ccrC1 expression in Staphylococcus haemolyticus NW19. Transcriptional levels of ccrC1, recA and lexA in NW19 with different antibiotic induction (1/8 MIC) and UV exposure. Gene expression levels were normalized and are presented relative to the trypticase soy broth (TSB) cultured control.

Figure 2.

Antibiotics promote SCC excision via ccrC1. Primer pairs corresponding to the two sides of SCCmec (EGRT-F and EGRT-R) were used to determine the ratio of the SCC-excised genome. The arrowheads indicate values below the limit of detection (<2.5 × 10⁻¹¹). One copy of the gene tpi (triosephosphate isomerase gene) was used as a reference. The bars represent the averages of three independent measurements in three different cultures, and the error bars represent the standard error of the mean. OXA, oxacillin; TEC, teicoplanin; VAN, vancomycin; GEN, gentamicin; KAN, kanamycin; TET, tetracycline; SMX, sulfamethoxazole; CIP, ciprofloxacin; TMP, trimethoprim.

Figure 3.

Alignment of the promoter regions of ccrC1 genes from Staphylococcus aureus WIS (accession number AB121219), S. aureus TSGH17 (accession number AB512767), S. aureus 85/2082 (accession number AB037671), S. aureus M (U10927), Staphylococcus haemolyticus JCSC1435 (AP006716), S. aureus PM1 (AB462393) and Staphylococcus saprophyticus ATCC15305 (AP008934). Type V(5C2&5) SCCmec in S. haemolyticus NW19 contains two ccrC-carrying gene complexes on either side of mec gene complex. The ccrC1 allele 8 is located in the region between orfX and mec gene complex, while ccrC1 allele 2 is located between mec gene complex and ydhK complex (as showed in Supplementary Figure S2A). Two allele genes were 92.5% identical. The putative LexA-binding sequences are underlined. Promoter elements (−35 and −10) are framed. The start codon of ccrC1 gene is indicated by a black arrow.

Figure 4.

(A) Partial sequences of the ccrC1 promoter region (wt) and SOS box mutants (mutA, mutB, mutC and mutBC) used in the electrophoretic mobility shift assays (EMSAs). (B) Western blotting detection of green fluorescent protein (GFP) under the wild-type and mutant promoters of ccrC1. The plasmids pCL::ccr-gfp and pCL::mutccr-gfp, which encode the wild-type and mutant promoters of ccrC1, respectively, followed by gfp, were transformed into NW19. Cells were grown until OD₆₀₀ = 0.6 and total bacterial protein was prepared. (C) EMSA experiments performed with the LexA protein and wild-type ccrC1 promoter. (D) EMSA of the wild-type or mutant ccrC1 promoter and LexA protein.

Figure 5.

Effects of recA and lexA on the expression of ccrC1 and SCC excision frequency in NW19. (A) Relative expression of ccrC1 in different strains compared to the control (wild-type without mitomycin C (MMC)). (B) SCC excision frequency detection in different strains with and without MMC treatment. (C) Detection of GFP using ccr-gfp reporter strategy. Wild, wild-type NW19; recDw, NW19 with reduced expression of recA; recUp, NW19 with increased expression of recA; lexDw, NW19 with reduced lexA expression; lexUp, lexA transcript increased NW19; MMC−, without MMC exposure; MMC+, with MMC exposure. Arrowhead, below the limit of detection (<2.5 × 10⁻¹¹). The relative expression of ccrC1 and the SCC excision frequency were detected with and without MMC exposure (0.5 mg/L). Each sample was determined in triplicate. The data represent the mean ± Standard Error Mean (SEM).

Figure 6.

The SOS response promotes the expression of ccrA and SCCmec excision in Mu50. (A) Alignment of the promoter regions of ccrA genes from Staphylococcus aureus Mu50 (accession number BA000017), S. aureus COL (CP000046), S. aureus N315 (BA000018), Staphylococcus pseudintermedius KM241 (AM904731), S. aureus 85/3907 (AB047089) and S. aureus HDE288 (AF411935). The putative LexA-binding sequences are underlined. Promoter elements (−35 and −10) are boxed. The 5΄ ATG of ccrA gene is indicated by a black arrow. (B) EMSA experiments performed with the LexA protein and ccrA promoter. (C) Relative expression of ccrA, recA and lexA in wild-type Mu50, the recA-deleted mutant (ΔrecA) and recA-complemented mutant (ΔrecA::precA) compared to the control (wild-type without MMC). *, not detectable. (D) SCCmec excision frequency detection in different strains with and without MMC treatment (0.5 mg/l). Each sample was determined in triplicate. Data are presented as the average ± SEM.

Figure 7.

Proposed model of the regulatory pathway by which the SOS response promotes SCCmec transfer. Intermediary molecules involved in SOS induction are shown. The mechanism by which SCCmec transfers among staphylococci remains elusive.