Mette Damkjær Bartels

Rapid Increase of Genetically Diverse Methicillin-Resistant Staphylococcus aureus, Copenhagen, Denmark

Community-onset MRSA with diverse genetic backgrounds is rapidly emerging in this previously low MRSA prevalence area.

A Common Variant of Staphylococcal Cassette Chromosome mec Type IVa in Isolates from Copenhagen, Denmark, Is Not Detected by the BD GeneOhm Methicillin-Resistant Staphylococcus aureus Assay

ABSTRACT Rapid tests for detection of methicillin-resistant Staphylococcus aureus (MRSA) carriage are important to limit the transmission of MRSA in the health care setting. We evaluated the performance of the BD GeneOhm MRSA real-time PCR assay using a diverse collection of MRSA isolates, mainly from Copenhagen, Denmark, but also including international isolates, e.g., USA100-1100. Pure cultures of 349 MRSA isolates representing variants of staphylococcal cassette chromosome mec (SCCmec) types I to V and 103 different staphylococcal protein A (spa) types were tested. In addition, 53 methicillin-susceptible Staphylococcus aureus isolates were included as negative controls. Forty-four MRSA isolates were undetectable; of these, 95% harbored SCCmec type IVa, and these included the most-common clone in Copenhagen, spa t024-sequence type 8-IVa. The false-negative MRSA isolates were tested with new primers (analyte-specific reagent [ASR] BD GeneOhm MRSA assay) supplied by Becton Dickinson (BD). The ASR BD GeneOhm MRSA assay detected 42 of the 44 isolates that were false negative in the BD GeneOhm MRSA assay. Combining the BD GeneOhm MRSA assay with the ASR BD GeneOhm MRSA assay greatly improved the results, with only two MRSA isolates being false negative. The BD GeneOhm MRSA assay alone is not adequate for MRSA detection in Copenhagen, Denmark, as more than one-third of our MRSA isolates would not be detected. We recommend that the BD GeneOhm MRSA assay be evaluated against the local MRSA diversity before being established as a standard assay, and due to the constant evolution of SCCmec cassettes, a continuous global surveillance is advisable in order to update the assay as necessary.

Environmental meticillin-resistant Staphylococcus aureus (MRSA) disinfection using dry-mist-generated hydrogen peroxide

Meticillin-resistant Staphylococcus aureus (MRSA) is a major problem in hospitals worldwide. Hand hygiene is recognised as crucial in limiting the spread of MRSA but less is known about the role of MRSA reservoirs in the inanimate hospital environment. We evaluated the effect of hydrogen peroxide vapour diffused by Sterinis((R)) against MRSA in two experimental hospital settings and in two field trials. Dipslides were used for MRSA detection and quantification before and after using the Sterinis disinfection process. In the first experimental hospital setting, four epidemic MRSA strains were placed at five locations and left for one week. All strains survived the week but not the disinfection process. In field trial one 14 upholstered chairs from a department with many MRSA positive patients were left for one month in a closed room prior to disinfection. MRSA was found on the upholstery of four of the 14 chairs. Three chairs became MRSA negative immediately after the disinfection, the fourth 24h later. The second field trial was in the private home of a MRSA positive family of four individuals. One location was found MRSA positive, remaining so after the Sterinis cycles. We found Sterinis to be effective against MRSA in the experimental hospital setting and upholstered chairs, but not in the private home of heavily colonised MRSA patients.

An Unexpected Location of the Arginine Catabolic Mobile Element (ACME) in a USA300-Related MRSA Strain

In methicillin resistant Staphylococcus aureus (MRSA), the arginine catabolic mobile element (ACME) was initially described in USA300 (t008-ST8) where it is located downstream of the staphylococcal cassette chromosome mec (SCCmec). A common health-care associated MRSA in Copenhagen, Denmark (t024-ST8) is clonally related to USA300 and is frequently PCR positive for the ACME specific arcA-gene. This study is the first to describe an ACME element upstream of the SCCmec in MRSA. By traditional SCCmec typing schemes, the SCCmec of t024-ST8 strain M1 carries SCCmec IVa, but full sequencing of the cassette revealed that the entire J3 region had no homology to published SCCmec IVa. Within the J3 region of M1 was a 1705 bp sequence only similar to a sequence in S. haemolyticus strain JCSC1435 and 2941 bps with no homology found in GenBank. In addition to the usual direct repeats (DR) at each extremity of SCCmec, M1 had two new DR between the orfX gene and the J3 region of the SCCmec. The region between the orfX DR (DR1) and DR2 contained the ccrAB4 genes. An ACME II-like element was located between DR2 and DR3. The entire 26,468 bp sequence between DR1 and DR3 was highly similar to parts of the ACME composite island of S. epidermidis strain ATCC12228. Sequencing of an ACME negative t024-ST8 strain (M299) showed that DR1 and the sequence between DR1 and DR3 was missing. The finding of a mobile ACME II-like element inserted downstream of orfX and upstream of SCCmec indicates a novel recombination between staphylococcal species.

Rise and subsequent decline of community-associated methicillin resistant Staphylococcus aureus ST30-IVc in Copenhagen, Denmark through an effective search and destroy policy.

The number of patients with community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) has increased rapidly in Copenhagen, Denmark since 2003. Patients with the typical Panton-Valentine leukocidin-positive CA-MRSA clone ST30-IVc were contacted with the aim of treating MRSA carriers, evaluating the effect of MRSA eradication therapy (ET), and finding links among patients. Twenty-three index patients infected with the ST30-IVc clone from November 2003 to September 2005 were contacted and transmission chains were studied. The majority of ST30-IVc patients had a connection to the Philippines. Household members were screened for MRSA and all members of families with MRSA carriers were offered treatment of the carrier state and were followed for 1 year. MRSA carriers were found in seven of 16 households and transmission occurred among close contacts and in kindergartens. Five days of ET was insufficient and at least one person in each household was treated with systemic antibiotics. All families were MRSA negative at 1-year follow-up. The CA-MRSA clone ST30-IVc has been imported to Copenhagen, Denmark, primarily from the Philippines, and has spread through close contacts and in kindergartens. Treatment of MRSA carriers was difficult and required many resources, but the clone was eventually successfully eliminated. The import of ST30-IVc to Denmark will continue, but the spread of the clone in Denmark can be kept to a minimum by direct intervention in the affected families.

Associations between dru Types and SCCmec Cassettes

Molecular typing is an important tool in the investigation of methicillin resistant Staphylococcus aureus (MRSA) outbreaks and in following the evolution of MRSA. The staphylococcal cassette chromosome mec (SCCmec) contains a hypervariable region with a variable number of 40 bp repeats named direct repeat units (dru). The dru region has been suggested as a supplementary typing method for MRSA and an international nomenclature exists. The purpose of this study was to investigate the diversity and variability of the dru region in a diverse collection of MRSA. We studied 302 MRSA isolates harbouring SCCmec types I to VI. The isolates represented a broad genetic background based on Staphylococcal protein A (spa) typing and multilocus sequence typing (MLST) and included 68 isolates (68 patients) from an outbreak with t024-ST8-IVa and 26 isolates from the same patient. Sequencing identified 53 dru types (dt) in 283 isolates, while eighteen isolates contained no dru repeats and one isolate resisted sequencing. The most common dru type, dt10a, was present in 53% of the sequenced isolates and was found in all SCCmec types, except type II. Seven (10%) of the 68 epidemiologically related patients had isolates with dru type variants indicating that dru typing is not useful as a first line epidemiological typing tool. However, MRSA isolates cultured from a single patient over a three year period exhibited a single dru type. The finding of dt10a in most SCCmec types suggests that dru and mecA originate from the same Staphylococcus species.

Whole Genome Sequencing of Danish Staphylococcus argenteus Reveals a Genetically Diverse Collection with Clear Separation from Staphylococcus aureus

Staphylococcus argenteus (S. argenteus) is a newly identified Staphylococcus species that has been misidentified as Staphylococcus aureus (S. aureus) and is clinically relevant. We identified 25 S. argenteus genomes in our collection of whole genome sequenced S. aureus. These genomes were compared to publicly available genomes and a phylogeny revealed seven clusters corresponding to seven clonal complexes. The genome of S. argenteus was found to be different from the genome of S. aureus and a core genome analysis showed that ~33% of the total gene pool was shared between the two species, at 90% homology level. An assessment of mobile elements shows flow of SCCmec cassettes, plasmids, phages, and pathogenicity islands, between S. argenteus and S. aureus. This dataset emphasizes that S. argenteus and S. aureus are two separate species that share genetic material.

Structural Variations of Staphylococcal Cassette Chromosome mec Type IVa in Staphylococcus aureus Clonal Complex 8 and Unrelated Lineages

ABSTRACT PCR mapping of staphylococcal cassette chromosome mec type IVa and adjacent mobile elements in 94 methicillin-resistant Staphylococcus aureus (MRSA) strains identified two primary structures (A and B) that could be further classified into two (A1 and A2) and five (B1 to B5) variants, primarily based on structural differences in the orfX-J3 region. While spa type t008 (USA300) invariably contained the A variants, other spa types belonging to clonal complex 8 and unrelated lineages generally contained B variants. These findings have important implications for the typing and identification of MRSA strains containing B variants.

Complete Genome Sequence of Staphylococcus aureus Strain M1, a Unique t024-ST8-IVa Danish Methicillin-Resistant S. aureus Clone

ABSTRACT We report the genome sequence, in five contigs, of a methicillin-resistant Staphylococcus aureus isolate designated M1. This clinical isolate was from the index patient of a methicillin-resistant Staphylococcus aureus (MRSA) outbreak in Copenhagen, Denmark, that started in 2003. This strain is sequence type 8 (ST8), spa type t024, and staphylococcal cassette chromosome mec element (SCCmec) type IVa.

Global spread of three multidrug-resistant lineages of Staphylococcus epidermidis

Staphylococcus epidermidis is a conspicuous member of the human microbiome, widely present on healthy skin. Here we show that S. epidermidis has also evolved to become a formidable nosocomial pathogen. Using genomics, we reveal that three multidrug-resistant, hospital-adapted lineages of S. epidermidis (two ST2 and one ST23) have emerged in recent decades and spread globally. These lineages are resistant to rifampicin through acquisition of specific rpoB mutations that have become fixed in the populations. Analysis of isolates from 96 institutions in 24 countries identified dual D471E and I527M RpoB substitutions to be the most common cause of rifampicin resistance in S. epidermidis, accounting for 86.6% of mutations. Furthermore, we reveal that the D471E and I527M combination occurs almost exclusively in isolates from the ST2 and ST23 lineages. By breaching lineage-specific DNA methylation restriction modification barriers and then performing site-specific mutagenesis, we show that these rpoB mutations not only confer rifampicin resistance, but also reduce susceptibility to the last-line glycopeptide antibiotics, vancomycin and teicoplanin. Our study has uncovered the previously unrecognized international spread of a near pan-drug-resistant opportunistic pathogen, identifiable by a rifampicin-resistant phenotype. It is possible that hospital practices, such as antibiotic monotherapy utilizing rifampicin-impregnated medical devices, have driven the evolution of this organism, once trivialized as a contaminant, towards potentially incurable infections.Genomic analysis uncovers global prevalence of three multidrug-resistant Staphylococcus epidermidis lineages encoding rifampicin resistance and reduced susceptibility to glycopeptide antibiotics.