Typing of staphylococcal SCCmec elements using DNA microarrays Stefan Monecke 1,2,3, Darius Gawlik 1,2,3, Elke Müller 1,3, Annett Reissig 1,3, Antje Ruppelt-Lorz 2, Peter Slickers 1,3, Ralf Ehricht 1,3 1
2
Alere Technologies GmbH, Jena, Germany Institute for Medical Microbiology and Hygiene, Technical University of Dresden, Dresden, Germany 3 InfectoGnostics Research Campus Jena, Jena, Germany
Introduction: SCCmec elements are mobile genetic elements in Staphylococci that carry methicillin resistance genes mecA or mecC in addition to recombinase genes and accessory genes. Twelve main types and several variants have been described so far 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. Accessory genes associated with SCCmec might include fusC (fusidic acid resistance), the ACME and kdp operons, as well as several genes coding for heavy metal resistances. These genes also can occur in SCC elements that lack mec genes allowing the assumption that SCC elements as a vehicle for horizontal gene transfer in Staphylococci predate the emergence of MRSA and that they also could transport other “payloads” than mecA/C genes. Materials and Methods: 85 markers were selected because of an unambiguous, strict linkage to SCC elements. Target genes are shown in Table 1. Various resistance markers known to be occasionally associated with SCC elements (such as aadD, erm(A) or tet genes) were also detected by the array, but they were excluded from analyses because hybridisation cannot provide information whether they were localised on SCC elements or, e.g., on plasmids. 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 was screened that consisted of about 1,200 isolates of S. aureus/MRSA and some coagulase-negatives. Clonal strains were cultured on Columbia blood agar plates, harvested and enzymatically lysed. After DNA preparation, a multiplexed linear amplification was performed using one specific primer per target. During amplification, biotin-16-dUTP was randomly incorporated into the amplicons. After incubation on the array and after washing steps, the hybridisation to the probes on the array was detected using streptavidin-horseradish-peroxidase that catalysed a localised dye precipitation, and thus a visible spot formation. Microarrays were then photographed and analysed. Results were used for constructing a database of both, experimentally observed and bioinformatically predicted SCC profiles, and they also allowed assignment to clonal complexes and strains.
Target gene
Explanation
Target gene
Explanation
Target gene
Explanation
Target gene
Explanation
Target gene
Explanation
adhC
Alcohol dehydrogenase, zinc-containing
cas1
(M06-0171)
CRISPR-assoc. endonuclease, from CC779 MRSA
D1GU38 (TW20)
Putative protein, allele from TW20
merB
Alkylmercury lyase
SCC terminus 03
SCC integration site alternate to dcs
arcA-SCC
Arginine deiminase
cas1
(MSHR1132)
CRISPR-assoc. endonuclease, from CC1850/argenteus
D1GU55
Putative membrane protein
mvaS -SCC
Truncated 3-hydroxy-3-methylglutaryl CoA synthase
SCC terminus 04
SCC integration site alternate to dcs
arcB-SCC
Ornithine carbamoyltransferase
ccrA-1
Cassette chromosome recombinase A, type 1
D3JD07
Putative protein
opp3B
Oligopeptide permease, channel-forming protein
SCC terminus 05
SCC integration site alternate to dcs
arcC-SCC
Carbamate kinase
ccrA-2
Cassette chromosome recombinase A, type 2
Delta mecR1
Truncated methicillin resistance operon repressor 1
opp3C
Oligopeptide permease, channel-forming protein
SCC terminus 06
SCC integration site alternate to dcs
arcD-SCC
Arginine/ornithine antiporter
ccrA-3
Cassette chromosome recombinase A, type 3
DUF1958
Putative protein
pls -SCC (CO L)
Plasmin-sensitive surface protein.
SCC terminus 07
SCC integration site alternate to dcs
arsB- SCC
Arsenical pump membrane protein (3 probes for 3 alleles)
ccrA-4
Cassette chromosome recombinase A, type 4
fusC/ Q6GD50
SCC-associated fusidic acid resistance gene
PSM-mec
Phenol soluble modulin from SCCmec
SCC terminus 09
SCC integration site alternate to dcs
arsC -SCC
Arsenate reductase
ccrAA
“Cassette chromosome recombinase AA”
kdpA -SCC
Potassium-translocating ATPase A, chain 2
Q3YK51
Putative protein
SCC terminus 10
SCC integration site alternate to dcs
B2Y834
Abortive phage resistance protein
ccrB-1
Cassette chromosome recombinase B, type 1
kdpB -SCC
Potassium-transporting ATPase B, chain 1
Q4LAG7 (consensus)
Putative protein from SCCmec type V/VT/SCCfus elements
SCC terminus 11
SCC integration site alternate to dcs
B6VQU0
Putative protein
ccrB-2
Cassette chromosome recombinase B, type 2
kdpC -SCC
Potassium-translocating ATPase C, chain 2
Q4LAG7 (SCCfus)
Putative protein from SCCfus elements
SCC terminus 12
SCC integration site alternate to dcs
blaZ
Beta-lactamase from SCCmec XI
ccrB-3
Cassette chromosome recombinase B, type 3
kdpD -SCC
Sensor kinase protein
Q4LAG7 (SO 385)
Putative protein from SCCmec type V/VT elements
SCC terminus 13
SCC integration site alternate to dcs
C5QAP8 (SCCmec XI) Putative protein
ccrB-4
Cassette chromosome recombinase B, type 4
kdpE -SCC
KDP operon transcriptional regulatory protein
Q8CU82
Putative protein
SCC terminus 14
SCC integration site alternate to dcs
cadD
(R35)
Cadmium transport protein D
ccrC
Cassette chromosome recombinase C
mco -SCC
Multi copper oxidase
Q933A2
Putative ADP-ribosyltransferase
speG
Spermidine N-acetyltransferase
cadX
(JCSC6943)
Putative regulator of cadmium efflux
copA2 -SCC
copper exporting ATPase
mecA
Modified penicillin binding protein (PBP2a)
Q93IB7
LytTR domain DNA-binding regulator
tirS
Staphylococcal TIR-protein binding protein
capH1
capsular polysaccharide biosynthesis protein Cap1H
cstB -SCC1/Q2G1R6 CsoR-like sulfur transferase-regulated gene, pseudogene
mecC
Alternate gene encoding a modified penicillin binding protein
Q9S0M4
Putative protein
ugpQ
Glycerophosphoryl diester phosphodiesterase
capI1
capsular polysaccharide biosynthesis protein Cap1I
cstB- SCC2/ Q2G1R6 CsoR-like sulfur transferase-regulated gene
mecI
Methicillin-resistance regulatory protein
Q9XB68-dcs
Located at the terminus of SCCmec directly next to orfX
xylR/mecR2
Methicillin resistance operon repressor 2, xylose repressor homolog
capJ1
capsular polysaccharide biosynthesis protein Cap1J
czrC
Cadmium/zinc resistance gene C, heavy metal translocating ATPase mecR1
Methicillin resistance operon repressor 1
SCC terminus 01
SCC integration site alternate to dcs
ydhK
Putative lipoprotein
capK1
capsular polysaccharide biosynthesis protein Cap1K
D1GU38
Putative protein
Mercury reductase
SCC terminus 02
SCC integration site alternate to dcs
yeeA
(FPR3757)
(SCCmec XI)
(MRSAZH47)
(85-2082)
merA
(FPR3757)
(FPR3757)
Putative DNA methyltransferase
Table 1: Target genes for subtyping SCC/SCCmec elements
Results: An extensive variety of SCCmec and other SCC elements was experimentally observed and/or bioinformatically predicted from published sequences. This includes currently 276 different SCCmec patterns, or variants thereof, harbouring mecA, three that harbour mecC, one that carries both, mecA and mecC (Staphylococcus sciuri carnaticus GVGS2, GenBank HG515014.1) as well as 78 ACME-, fusC- and other SCC elements without mecA/C genes. Interestingly, some widespread “strains” engulf several variants with distinct SCCmec subtypes. Notable examples are ST239-MRSA-III (with currently 24 distinguishable variants among 137 tested isolates), CC22-MRSA-IV (22 variants in 153 isolates), or CC398-MRSA-V (13 variants in 100 isolates). The “true” number of variants can be expected to be even higher, as hybridisation cannot recognise minor sequence polymorphisms, gene duplications or inversions.
Sometimes, SCC subtypes correlate with geographical origin. This was for instance the case in PVL-positive CC8-MRSA-IV, where USA300 from the USA, Germany or Australia carried SCCmec IVa+ACME1+copA2 (copper resistance) while isolates from Latin America and Spain had SCCmec IVc+merA/B+copA2+mco (mercury and copper resistance). Similarly, CC22-MRSA-IV/”EMRSA-15” from Western Europe and the Gulf states carry SCCmec IVh/j while otherwise identical isolates from the Kingdom of Saudi Arabia harboured an SCCmec IVa variant suggesting that they emerged independently (probably from the Middle Eastern “Gaza Clone”, by loss of tst1). < Figure 1: A network tree showing similarities (although not necessarily phylogenetic relationships) of different SCCmec hybridisation patterns. This includes both, experimentally identified types as well as predictions from publicly accessible genome sequences. SCC elements without mec genes are not included.
Clonal complex
Number of SCCmec variants observed in this CC
SCCmec subtype
n
SCCmec IVa (MW2)
19
Clonal complexes in which that particular subtype was observed
CC5
58
CC8
48
CC22
25
CC8 (ST239)
24
CC398
21
CC30
16
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).
CC45 [agr I]
15
SCCmec IVb/d/i (JCSC1978/6668/4469)
10
CC5, CC7, CC8, CC22, CC30, CC45 [agr I], CC59, CC88, CC97, S. argenteus CC2198
CC45 [agr IV-cap 8]
11
SCCmec VT (PM1)
8
CC5, CC6, CC20, CC30, CC45 [agr I], CC59, CC97, CC509
CC1, CC1 (ST573/772), CC8 (ST72), CC88
10
SCC [mec VT+czrC ] (SO385)
7
CC1, CC1 (ST573/772), CC8, CC30, CC45 [agr IV-cap 8], CC398, CC2817
CC59, CC97
9
SCCmec XI (LGA251/M10-61)
6
CC49, CC130, CC425, CC599, CC1943, ST2616
CC7, CC361
5
SCCmec IVc (IS-105)
6
CC5, CC8, CC8 (ST72), CC22, CC45 [agr I], CC80
CC779
4
SCCmec IVa (CMFT503)
6
CC5, CC8, CC22, CC88, CC121, CC398
CC9, CC80, CC121
3
SCCmec IVa (H131520133)
5
CC5, CC30, CC59, CC88, CC152
SCCmec IVg (SA40)
4
CC59, CC96, ST140, CC361
SCC [mec VT+fus ] (Unknown, CC5/ST72)
4
CC5, CC8 (ST72), CC59, CC361
SCC [mec IV+fus+tir ] (CC22/CC45)
4
CC5, CC22, CC30, CC45 [agr I]
Table 2 : Number of different SCCmec patterns pro CC.
Table 3 : The most widespread SCCmec patterns and the clonal complexes in which they can be found.
SCC elements that carry mecA are shown in red, those with mecC in blue; and the one with both, in purple. Black labels describe specific SCCmec elements from well known reference strains; grey labels indicate groups or clusters of similar variants.
The highest numbers of different patterns were observed in CC5 and CC8 suggesting that these CCs are especially „promiscuous“ with regard to uptake of foreign mobile genetic elements. The number of SCC variants pro CC is not a function of its mere abundance. For instance, CC15 is one of the most common lineages. Nevertheless only a single type of SCCmec was found in about 1,200 isolates screened.
This table shows the most widespread SCCmec variants, with regard to the number of clonal complexes in which they were yet detected. This shows that the most mobile elements all belong to SCCmec IV, VT and, surprisingly XI. For comparison, COL-like SCCmec I has been found only in two CCs (CC5 and CC8) while SCCmec II (N315) was identified in CC5 and CC30 or SCCmec II (JH1/JH9) in CC5, CC8 and CC45.
CC6, CC9 (ST834), CC12, CC49, CC50, CC96, CC152, CC188, CC445, CC509, CC599, CC913, S. argenteus CC2250 Another 19 S. aureus and 4 S. argenteus CCs
2 1
Discussion: The high number of different SCC-associated hybridisation patterns and their appearance across strains and species indicate both, a rapid and ongoing evolution of SCCmec as well as a very high rate of transmissions within S. aureus and across staphylococcal species. The variability of SCCmec elements allows to distinguish isolates that appear similar or identical by other typing methods and this could be helpful for epidemiological typing. The microarray described herein can be used as high-throughput screening tool for the detection of novel SCC variants that warrant detailed investigation and sequence analysis.
CC1, CC5, CC6, CC7, CC8, CC8 (ST72), CC9, CC22, CC30, CC45 [agr I], CC45 (ST617), CC59, CC88, ST93, CC97, CC188, CC398, CC509, S. argenteus CC1223
SCCmec IVc (TCH60)
14
CC5, CC8, CC8 (ST72), CC9 (ST834), CC12, CC22, CC30, CC45 [agr I], CC59, CC80, CC88, CC97, CC913, S. argenteus CC2596
SCCmec VT (GR1)
12
CC1 (ST573/772), CC5, CC8 (ST72), CC30, CC45 [agr I], CC45 [agr IV-cap 8], CC88, CC121, CC152, CC361, CC398, ST2816
Both tables also show that the newer SCCmec IV and VT elements by now have spilled over into exotic and obscure lineages such as CC361, CC509, ST2816 or Staph. argenteus, indicating that MRSA became a truly ubiquitous and pandemic issue.
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