Extensive Diversity of SCCmec Elements in Staphylococci Stefan Monecke1,2,3, Darius Gawlik3,4, Elke Müller1,2, Annett Reissig1,2, Antje Ruppelt-Lorz3, Peter Slickers1,2, Ralf Ehricht1,2 1 Alere
Technologies GmbH, Jena 2 InfectoGnostics Research Campus, Jena 3 Institute of Medical Microbiology and Hygiene, Technische Universität Dresden 4 Hochschule Hamm-Lippstadt
Background: SCCmec elements are mobile genetic elements in different species of Staphylococci that carry methicillin resistance genes, recombinase and accessory genes. Twelve main types and several variants have been described based on the identity of the mec complex (i.e., mecA/C, regulatory genes and associated insertion sequences) and the alleles of the SCC associated recombinase genes (http://www.sccmec.org). In addition, there are also other SCC elements harbouring fusC (encoding fusidic acid resistance), ACME, heavy metal resistance or other genes allowing the assumption that SCC elements as a vehicle for horizontal gene transfer in Staphylococci predate the emergence of methicillin resistance and that they also could transport other “payloads” than mecA/C genes. Methods: 81 markers were selected from published SCC sequences because of an unambiguous, strict linkage to SCC elements and their variable presence in those elements. Various resistance markers known to be occasionally associated with SCC elements (such as aadD, erm(A) or tet genes) were detected, but excluded from analyses because hybridisation cannot provide information whether they were associated with SCC elements or, e.g., plasmids. Target genes are shown in Table 1. Hybridisation patterns for about 390 published genome or SCC sequences were theoretically predicted and categorised into subtypes. For practical validation and protocol optimisation, experiments with known reference strains were performed stepwise modifying hybridisation and washing temperatures until experiments yielded results that were in accordance to the theoretical predictions. Then, a strain collection of about 1,000 isolates of S. aureus/MRSA as well as of other staphylococcal species was screened. Strains were cultured and cloned on Columbia blood agar plates, harvested and enzymatically lysed. After DNA preparation, a multiplexed linear amplification was performed using one specific primer per target. Biotin-16-dUTP was randomly incorporated into the amplicons during that step. After incubation with the array and after washing steps, the hybridisation to the probes on the array was detected using streptavidin-horseradish-peroxidase that catalyses a local precipitation of a dye, and thus a spot formation. Microarrays were then photographed and analysed resulting in a database of both, experimentally observed and bioinformatically predicted SCC profiles.
Target gene mecA mecC adhC (FPR3757) arcA- SCC arcB- SCC arcC- SCC arcD- SCC arsB- SCC arsC- SCC B2Y834 B6VQU0
Explanation Modified penicillin binding protein (PBP2a) Alternate gene for modified penicillin binding protein Alcohol dehydrogenase, zinc-containing Arginine deiminase Ornithine carbamoyltransferase Carbamate kinase Arginine/ornithine antiporter Arsenical pump membrane protein Arsenate reductase Abortive phage resistance protein Putative protein
Target gene ccrA-2 ccrA-3 ccrA-4 ccrAA ccrB-1 ccrB-2 ccrB-3 ccrB-4 ccrC (85-2082) copA2- SCC cstB- SCC1 cstB -SCC2 czrC D1GU38
blaZ
Beta-lactamase
D1GU55
Putative membrane protein
pls- SCC
Putative protein
D3JD07
Putative protein
PSM-mec
Phenol soluble modulin from SCCmec
SCC terminus 09
Cadmium transport protein D Putative regulator of cadmium efflux Locus encoding SCC associated capsule type 1 CRISPR-associated endonuclease 1 Cassette chromosome recombinase A, type 1
Delta mec R1 DUF1958 fusC ( Q6GD50) kdpA- SCC kdpB- SCC
Truncated methicillin resistance operon repressor 1 Putative protein SCC-associated fusidic acid resistance gene Potassium-translocating ATPase A, chain 2 Potassium-transporting ATPase B, chain 1
Q3YK51 Q4LAG7 Q8CU82 Q933A2 Q93IB7
Putative protein Putative protein from SCCmec type V/SCCfus elements Putative protein Putative ADP-ribosyltransferase LytTR domain DNA-binding regulator
SCC terminus 10 SCC terminus 11 SCC terminus 12 SCC terminus 13 SCC terminus 14
Table 1: Targets used for SCC typing (publication submitted):
(SCCmec XI)
C5QAP8 (SCCmec cadD (R35) cadX (JCSC6943) cap 1 cas1 (M06-0171) ccrA-1
XI)
Explanation Target gene Cassette chromosome recombinase A, type 2 kdpC- SCC Cassette chromosome recombinase A, type 3 kdpD- SCC Cassette chromosome recombinase A, type 4 kdpE- SCC “Cassette chromosome recombinase AA” mco- SCC Cassette chromosome recombinase B, type 1 mecI Cassette chromosome recombinase B, type 2 mec R1 Cassette chromosome recombinase B, type 3 merA Cassette chromosome recombinase B, type 4 merB Cassette chromosome recombinase C mvaS- SCC Copper exporting ATPase opp3B, CsoR-like sulfur transferase-regulated gene B, SCCmec II opp3B (C427) CsoR-like sulfur transferase-reg. gene B, SCCmec IVa opp3B (FPR3757) Cadmium and zinc resistance gene C opp3C (C427) Putative protein opp3C (FPR3757)
Explanation Potassium-translocating ATPase C, chain 2 Sensor kinase protein KDP operon transcriptional regulatory protein Multi copper oxidase Methicillin-resistance regulatory protein Methicillin resistance operon repressor 1 Mercury reductase Alkylmercury lyase Truncated 3-hydroxy-3-methylglutaryl CoA synthase
(COL)
Explanation Putative protein Spermidine N-acetyltransferase Staphylococcal TIR-protein binding protein Glycerophosphoryl diester phosphodiesterase Methicillin resist. operon repressor 2 Putative lipoprotein Putative DNA methyltransferase Located at the terminus of SCCmec directly next to orfX.
Oligopeptide permease, channel-forming protein Oligopeptide permease, USA300 allele
Target gene Q9S0M4 speG ( FPR3757) tirS ugpQ xylR/mecR2 ydhK (FPR3757) yeeA Q9XB68-dcs SCC terminus 01 SCC terminus 02 SCC terminus 03 SCC terminus 04 SCC terminus 05 SCC terminus 06
Plasmin-sensitive surface protein
SCC terminus 07
SCC integration sites alternate to dcs
Oligopeptide permease, channel-forming protein
Results: Altogether, 314 different SCC patterns were discerned either by in vitro hybridisation experiments, or by in silico analysis of published sequences, or by both approaches combined. The “true number” of types/subtypes still could be different, because i) hybridisations cannot detect inversions or duplications of genes and ii) some markers might also be situated outside of SCC elements although for the majority of the selected markers (except merA/B) this has not yet been observed. A wide variety of both, known and unknown patterns of potentially new types, subtypes, pseudoSCC elements and composite elements was observed and analysed (Figure 1).
Figure 1: A network tree showing similarities (although not necessarily phylogenetic relationships) of 314 different SCC hybridisation patterns. This includes both, experimentally identified types as well as predictions from publicly accessible genome sequences. Array hybridisation profiles or predicted patterns were converted into a series of ‘sequences’. Each position in these ‘sequences’, i.e., each probe, could have a value of ‘positive’ (‘C’), ‘negative’ (‘G’) or ‘ambiguous’ (‘A’). These ‘sequences’ were used with SplitsTree version 4.11.3 (Huson DH, Bryant D; 2006) on default settings (characters transformation: uncorrected P/ignore ambiguous states, distance transformation: Neighbour-Net, and variance: ordinary least squares). SCC elements that carry mecA are shown in red, those with mecC in blue; and those which lack mec genes are grey. The latter include SCCfus, ACME, heavy metal resistance and other elements.
The lineages with the highest diversities of SCC elements were CC5 and CC8 (the latter in a narrow sense: ST72 and ST239 being analysed separately) for which 53 and 49 distinct SCC patterns were observed. On the other hand, common and pandemic lineages CC15 and CC121 presented with one and, respectively, two SCCmec elements only. Interestingly, some widespread epidemic “strains” were found to engulf distinct variants with different SCCmec subtypes. Notable examples are ST239-MRSA-III (with currently 22 distinguishable variants, see Table 2), CC22-MRSA-IV (17 variants), or CC398-MRSA-V (12 variants). Table 2: Twenty-two different SCCmec III variants from ST239-MRSA-III (most genes that were either always present or always absent are omitted from the table for the sake of clarity). Additional variants were found in CC5 and in coagulase-negatives. A “plain“ SCCmec III element without additional recombinase or heavy metal resistance genes was not identified in S. aureus but in S. pseudintermedius (such as KM1381, GenBank AM904732.1).
Identical SCC elements were observed in different strains and even species. The most widespread element seems to be the SCCmec IVa variant corresponding to MW2, GenBank BA000033.2. It was identified in as much as 19 distinct S. aureus lineages (CC1, CC5, CC6, CC7, CC8, CC9, CC22, CC30, CC45 [agr I], CC45 [agr IV-cap8], CC45 [ST617], CC59, CC72, CC88, ST93, CC97, CC188, CC398, CC509), in two S. argenteus lineages (CC1223, CC1850) and in S. pseudintermedius.
Conclusions: The proposed microarray can help to distinguish isolates that appear similar or identical by other typing methods and it can be used as highthroughput screening tool for the detection of possibly new SCC variants that warrant detailed investigation and sequence analysis. The high degree of diversity of SCC elements even within so-called strains could be helpful for epidemiological typing and outbreak investigations, especially with regard to widespread, common and otherwise homogenous strains. The wide spread across lineages and species of some elements indicates frequent transmission or recombination events, although the “susceptibility” of lineages to the acquisition of SCC elements varies. The diversity of elements within “one strain” (see Table 2) shows a further evolution and diversification once a transmission occurred. This raises the question on scale and actual speed of the evolution of SCC elements that undergo their own evolution as “selfish replicators” rather independently from their staphylococcal hosts they reside in. Contact:
[email protected],
[email protected]
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