This site needs JavaScript to work properly. Please enable it to take advantage of the complete set of features!
Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

NIH NLM Logo
Log in
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2014 Jun 27;9(6):e101419.
doi: 10.1371/journal.pone.0101419. eCollection 2014.

Recombinations in staphylococcal cassette chromosome mec elements compromise the molecular detection of methicillin resistance in Staphylococcus aureus

Affiliations

Recombinations in staphylococcal cassette chromosome mec elements compromise the molecular detection of methicillin resistance in Staphylococcus aureus

Grant A Hill-Cawthorne et al. PLoS One. .

Abstract

Clinical laboratories are increasingly using molecular tests for methicillin-resistant Staphylococcus aureus (MRSA) screening. However, primers have to be targeted to a variable chromosomal region, the staphylococcal cassette chromosome mec (SCCmec). We initially screened 726 MRSA isolates from a single UK hospital trust by recombinase polymerase amplification (RPA), a novel, isothermal alternative to PCR. Undetected isolates were further characterised using multilocus sequence, spa typing and whole genome sequencing. 96% of our tested phenotypically MRSA isolates contained one of the six orfX-SCCmec junctions our RPA test and commercially available molecular tests target. However 30 isolates could not be detected. Sequencing of 24 of these isolates demonstrated recombinations within the SCCmec element with novel insertions that interfered with the RPA, preventing identification as MRSA. This result suggests that clinical laboratories cannot rely solely upon molecular assays to reliably detect all methicillin-resistance. The presence of significant recombinations in the SCCmec element, where the majority of assays target their primers, suggests that there will continue to be isolates that escape identification. We caution that dependence on amplification-based molecular assays will continue to result in failure to diagnose a small proportion (∼4%) of MRSA isolates, unless the true level of SCCmec natural diversity is determined by whole genome sequencing of a large collection of MRSA isolates.

PubMed Disclaimer

Conflict of interest statement

Competing Interests: Olaf Piepenburg and Matthew S. Forrest are employed by TwistDx, and have a stake in the success of its technology. TwistDx is one of the funders of this study and is a UK based company, with a commercial interest in the use of the RPA technology for the detection of hospital-acquired pathogens. TwistDx Ltd, is the developer and manufacturer of RPA technology and is part of the of the Alere group of companies. Lyndsey O. Hudson was the recipient of a BBSRC CASE-funded PhD studentship from TwistDx. This work was also supported by Smart Solutions for HCAI. There are no further patents, products in development or marketed products to declare. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors.

Figures

Figure 1
Figure 1. Level of homology between 24 sequenced SCCmec elements using the Circos tool .
All-against-all BLASTN using E value of 10−300 as cut-off. 4738 local alignments produced in total, internal ribbons show 2465 alignments to preserve clarity. Histograms around circumference of circle show distribution of all 4738 alignments. Colours correspond to SCCmec type: red, IVh-CMFT3119; light purple, IVj; blue, IVa; purple, IVk; orange, IIa-CMFT2-like; yellow, IIa-CMFT492-like; dark orange, II.5; dark red, IVe; black, XI; green, V. Very little homology seen for CMFT540 (type XI) and region of CMFT3119 containing arc gene complex and ccrC.
Figure 2
Figure 2. Variant SCCmec elements.
a) SCCmec for archetypal type II, MRSA252 (NC_002952), compared to isolates CMFT2 and CMFT492. The cassette of CMFT2 shows the typical features of a type II SCCmec; a class A mec complex and a class 2 ccr complex. However, there is an additional SCC carrying type I ccr genes situated at the 5′ end of the element. CMFT492 is superficially similar and contains the same additional class 1 ccr complex. However, it lacks two of the major features of most type II SCCmec elements; the plasmid vector pUB110 and transposon Tn554. b) SCCmec type IVk: SCCmec for CMFT201 compared to CA05 (AB063172, type IV(2B)) and 45394F (GU122149). The cassette of CMFT201 shows the typical features of a type IV SCCmec (CA05); a class B mec complex and a class 2 ccr complex. However, there are additional SCC elements with an SCC carrying type I ccr genes situated at the 5′ end of the cassette. This is a similar structure to that shown by strain 45394F (unpublished). c) Type IVh variant SCC-SCCmec element for CMFT3119 compared to strains ZH47 (AM292304) and M1 (HM030720). The cassette of CMFT3119 shows the typical features of a type IV SCCmec; a class B mec complex and a class 2 ccr complex. However CMFT3119 contains an additional SCC carrying a ccrC gene upstream of the mec complex, similar to the recombination seen in ZH47. In contrast to ZH47 there is not a Tn4001 but instead part of the arginine catabolic mobile element (ACME), seen in S. epidermidis, S. haemolyticus and USA300, has been inserted. This arc gene cluster is very similar to that seen in the recently identified strain M1.

References

    1. Lowy FD (1998) Staphylococcus aureus infections. N Engl J Med 339: 520–532 10.1056/NEJM199808203390806 - DOI - PubMed
    1. Tsubakishita S, Kuwahara-Arai K, Sasaki T, Hiramatsu K (2010) Origin and molecular evolution of the determinant of methicillin resistance in staphylococci. Antimicrob Agents Chemother 54: 4352–4359 10.1128/AAC.00356-10 - DOI - PMC - PubMed
    1. Barbier F, Ruppé E, Hernandez D, Lebeaux D, Francois P, et al. (2010) Methicillin-resistant coagulase-negative staphylococci in the community: high homology of SCCmec IVa between Staphylococcus epidermidis and major clones of methicillin-resistant Staphylococcus aureus . J Infect Dis 202: 270–281 10.1086/653483 - DOI - PubMed
    1. Huletsky A, Giroux R, Rossbach V, Gagnon M, Vaillancourt M, et al. (2004) New real-time PCR assay for rapid detection of methicillin-resistant Staphylococcus aureus directly from specimens containing a mixture of staphylococci. J Clin Microbiol 42: 1875–1884. - PMC - PubMed
    1. Polisena J, Chen S, Cimon K, McGill S, Forward K, et al. (2011) Clinical effectiveness of rapid tests for methicillin resistant Staphylococcus aureus (MRSA) in hospitalized patients: A systematic review. BMC Infect Dis 11: 336 10.1186/1471-2334-11-336 - DOI - PMC - PubMed

Publication types

MeSH terms

Full text links
Public Library of Science full text link Public Library of Science Free PMC article
Cite

AltStyle によって変換されたページ (->オリジナル) /