Carriage of methicillin-resistant Staphylococcus aureus in children <6 years old: a retrospective follow-up study of the natural course and effectiveness of decolonization treatment.

BACKGROUND: Decolonization treatment of MRSA carriers is recommended in Denmark, except in households with MRSA-positive children <2 years old (wait-and-see approach). OBJECTIVES: To investigate a wait-and-see approach in children 2-5 years old, and the effect of decolonization treatment of MRSA carriage in all children <6 years old. PATIENTS AND METHODS: In this retrospective follow-up study, we included MRSA carriers <6 years old in the Capital Region of Denmark from 2007 to 2021. Data were collected from laboratory information systems and electronic patient records. We divided children into age groups of <2 years or 2-5 years and decolonization treatment versus no treatment. Treatment was chlorhexidine body washes and nasal mupirocin, sometimes supplemented with systemic antibiotics. Children were followed until becoming MRSA free, or censoring. The probability of becoming MRSA free was investigated with Cox regression (higher HRs indicate faster decolonization). RESULTS: Of 348 included children, 226 were <2 years old [56/226 (25%) received treatment] and 122 were 2-5 years old [90/122 (74%) received treatment]. Multivariable analyses did not show a larger effect of decolonization treatment versus no treatment in <2-year-olds (HR 0.92, 95% CI 0.52-1.65) or 2-5-year-olds (HR 0.54, 95% CI 0.26-1.12). Without treatment, 2-5-year-olds tended to clear MRSA faster than <2-year-olds (HR 1.81, 95% CI 0.98-3.37). CONCLUSIONS: We did not find a larger effect of decolonization treatment versus no treatment in children <6 years old, and 2-5-year-olds tended to become MRSA free faster than <2-year-olds. These results support a wait-and-see approach for all children <6 years old, but further studies are needed.

Within-host genomic evolution of methicillin-resistant Staphylococcus aureus in long-term carriers.

Assessing the genomic evolution of Staphylococcus aureus can help us understand how the bacteria adapt to its environment. In this study, we aimed to assess the mutation rate within 144 methicillin-resistant Staphylococcus aureus (MRSA) carriers with a carriage time from 4 to 11 years, including some carriers who belonged to the same households. We found that 23 of the 144 individuals had completely different MRSA types over time and were therefore not long-term carriers of the same MRSA. From the remaining 121 individuals, we performed whole-genome sequencing (WGS) on 424 isolates and then compared these pairwise using core genome multilocus sequence typing (cgMLST) and single-nucleotide polymorphism (SNP) analyses. We found a median within-host mutation rate in long-term MRSA carriers of 4.9 (3.4-6.9) SNPs/genome/year and 2.7 (1.8-4.2) allelic differences/genome/year, when excluding presumed recombination. Furthermore, we stratified the cohort into subgroups and found no significant difference between the median mutation rate of members of households, individuals with presumed continued exposure, e.g., from travel and persons without known continued exposure. Finally, we found that SNPs occurred at random within the genes in our cohort. KEY POINTS: • Median mutation rate within long-term MRSA carriers of 4.9 (3.4-6.9) SNPs/genome/year • Similar median mutation rates in subgroups (households, travelers) • No hotspots for SNPs within the genome.

Whole genome sequencing for surveillance of bacterial infectious illnesses.

This review summarises the current knowledge of whole genome sequencing (WGS) which has become the standard method for genetic characterisation of bacteria in surveillance and outbreak investigation. Although the method offers many advantages, its use in outbreak investigations is hampered by the relatively slow turn-around time. Using new approaches to perform WGS, typing and gene detection can now be completed within one day. This break-through allows clinical consequences to be taken almost immediately after detection of relevant bacteria and may have a huge impact on the future prevention of transmission of infectious diseases.

Whole genome sequencing reveals two genetically distinct MRSA outbreaks among people who inject drugs and homeless people in Copenhagen.

Whole genome sequencing (WGS) has greatly improved the detection of methicillin-resistant Staphylococcus aureus (MRSA) transmission between people. We describe the transmission of two unique MRSA clones among homeless people in Copenhagen using WGS and core genome MLST (cgMLST). In 2014, an accumulation of MRSA bacteremia cases among homeless people admitted to our hospital was recognized, all having the rare MRSA spa t5147/ST88. The European Typology of Homelessness and Housing Exclusion (ETHOS) categories revealed that people who inject drugs (PWID) frequently visiting the milieu but living in private accommodation accounted for most cases. Hoping to terminate the transmission, 161 homeless people were MRSA screened in 2015, but no additional cases were found. From 2009 to 2018, 60 patients with genomically related t5147/ST88 isolates were found, of these 70% were confirmed to come from the homeless setting and 17% had bacteremia. From 2017 to 2020, cgMLST revealed a smaller MRSA outbreak including 13 PWID with a completely different clone, t1476/ST8, of which 15% had bacteremia. Our study confirms that WGS and cgMLST is excellent to reveal MRSA outbreaks. The ETHOS categorization can be useful to find the primary source of spread in the homeless community.

spa Typing of Methicillin-Resistant Staphylococcus aureus Based on Whole-Genome Sequencing: the Impact of the Assembler.

Sequencing of the spa gene of methicillin-resistant Staphylococcus aureus (MRSA) is used for assigning spa types to e.g., detect transmission and control outbreaks. Traditionally, spa typing is performed by Sanger sequencing but has in recent years been replaced by whole-genome sequencing (WGS) in some laboratories. Spa typing by WGS involves de novo assembly of millions of short sequencing reads into larger contiguous sequences, from which the spa type is then determined. The choice of assembly program therefore potentially impacts the spa typing result. In this study, WGS of 1,754 MRSA isolates was followed by de novo assembly using the assembly programs SPAdes (with two different sets of parameters) and SKESA. The spa types were assigned and compared to the spa types obtained by Sanger sequencing, regarding the latter as the correct spa types. SPAdes with the two different settings resulted in assembly of the correct spa type for 84.8% and 97.6% of the isolates, respectively, while SKESA assembled the correct spa type in 98.6% of cases. The misassembled spa types were generally two spa repeats shorter than the correct spa type and mainly included spa types with repetition of the same repeats. WGS-based spa typing is thus very accurate compared to Sanger sequencing, when the best assembly program for this purpose is used. IMPORTANCE spa typing of methicillin-resistant Staphylococcus aureus (MRSA) is widely used by clinicians, infection control workers, and researchers both in local outbreak investigations and as an easy way to communicate and compare MRSA types between laboratories and countries. Traditionally, spa types are determined by Sanger sequencing, but in recent years a whole-genome sequencing (WGS)-based approach has become increasingly used. In this study, we compared spa typing by WGS using different methods for assembling the genome from short sequencing reads and compared to Sanger sequencing as the gold standard. We find substantial differences in correct assembly of spa types between the assembly methods. Our findings are therefore important for the quality of WGS based spa typing data being exchanged by clinical microbiology laboratories.

Estimated Roles of the Carrier and the Bacterial Strain When Methicillin-Resistant Staphylococcus aureus Decolonization Fails: a Case-Control Study.

Methicillin-resistant Staphylococcus aureus (MRSA) is a common bacterial pathogen that frequently colonizes healthy individuals, with potential to cause invasive infection. In Denmark, to keep the prevalence low, MRSA carriers are recommended to undergo decolonization treatments, but achieving decolonization is challenging. Knowledge about the factors contributing to decolonization is scarce. We aimed to identify bacterial genome and clinical factors influencing MRSA decolonization. We identified all new MRSA patients above 2 years of age within the Hvidovre catchment area, Copenhagen, Denmark, in 2017 and 2018. Carriers were defined as chronic carriers (cases) if they were MRSA positive after two or more treatments and as nonchronic carriers (controls) if they were MRSA free after the first or second treatment. Using whole-genome sequencing (WGS), we constructed a pangenome of bacterial strains. With the incorporation of bacterial genome and clinical patient data, machine learning and multivariate analyses were performed to determine the factors associated with decolonization. A total of 477 MRSA carriers were included. An age of ≥13 years was significantly associated with nonchronic carriage. We identified 278 bacterial genetic features that were statistically significantly associated with chronic carriage (P < 0.05 by Fisher's exact test). Chronic MRSA carriage was predicted with 68% accuracy using a combination of bacterial genome data and patient clinical data. Decolonization success is multifactorial. Apart from the 68% predicted accuracy found in this study, we estimate that the remaining 32% is a result of host factors and microbiome composition. IMPORTANCE Carriage of methicillin-resistant Staphylococcus aureus (MRSA) and other multiresistant bacteria is a prerequisite for infection and transmission. Successful decolonization treatment removes these risks. We aimed to identify bacterial genome and host clinical factors that influence MRSA decolonization to estimate the roles of the carrier and the bacterial strain, respectively, when decolonization fails. The long-term goal, beyond this study, is to optimize decolonization success, minimize MRSA transmission, and, ultimately, improve the quality of life of MRSA carriers.

A randomized, placebo-controlled, double-blinded trial of MRSA throat carriage treatment, with either standard decolonization alone or in combination with oral clindamycin.

BACKGROUND: Staphylococcus aureus is a frequent colonizer of the human skin and mucous membranes but can also cause a variety of serious infections. Antimicrobial resistance is an increasing worldwide challenge and is mainly driven by an overuse of antimicrobials. To avoid the spread of methicillin-resistant Staphylococcus aureus (MRSA) in Denmark, the Danish Health Authority recommends decolonization treatment of MRSA carriers and their household contacts. Standard decolonization treatment includes chlorhexidine body wash and mupirocin nasal ointment, especially throat carriage is difficult to treat. The broad-spectrum antibiotic, clindamycin, is often added to the decolonization treatment, but there is currently low scientific evidence for this treatment. AIM: To investigate whether the addition of clindamycin to the standard decolonization treatment increases decolonization success in MRSA throat carriers. METHODS: A randomized, placebo-controlled, double-blinded trial, including patients ≥ 18 years, who tested MRSA positive in the throat after completing one standard decolonization treatment. All carriers included in the trial receive standard decolonization treatment and are randomized to treatment with either placebo or clindamycin capsules for 10 days. We plan to include 40 participants in each of the two treatment arms. DISCUSSION: Due to the lack of consistent scientific evidence of clindamycin's effect in MRSA decolonization and the worldwide urgent need to reduce the use of antibiotics, we judged that a 30% increase in the decolonization success rate in carriers treated with clindamycin is appropriate to justify prescribing clindamycin as part of the decolonization treatment of asymptomatic MRSA carriers. TRIAL REGISTRATION: EudraCT number 2019-002631-29.

The use of core genome multilocus sequence typing to determine the duration of vancomycin-resistant Enterococcus faecium outbreaks.

The prevalence of vancomycin-resistant Enterococcus faecium has increased rapidly, and in Denmark, we are facing an endemic outbreak situation in hospitals. The aim of this study was to use whole-genome sequencing (WGS) and core genome multilocus sequencing typing (cgMLST) to determine the duration of VREfm outbreaks and thereby evaluate the effect of our infection control strategies. We included all VREfm isolates from six hospitals in the Capital Region of Denmark that were sequenced between 2012 and 2020. Ward data were collected from our laboratory information system. A ward outbreak was defined as two patient samples from the same ward within a period of 30 days belonging to the same cgMLST cluster. cgMLST complex types were determined using Ridom SeqSphere v7.2.3, where a maximum of 20 allelic differences between isolates defines a cluster. We included 1690 patient isolates between 2012 and 2020. Our collection consisted of 45 unique clusters and 227 ward outbreaks. The median duration of outbreaks was 20 days. We reported a median outbreak duration of VREfm outbreaks based on WGS data to be 20 days, and thus concluded that our infection control precautions are adequate.

Substantial Decrease in Vancomycin-Resistant Enterococcus faecium Outbreak Duration and Number of Patients During the Danish COVID-19 Lockdown: A Prospective Observational Study.

Vancomycin-resistant Enterococcus faecium (VREfm) is a globally significant nosocomial pathogen with a rapidly increasing prevalence. The objectives were to investigate VREfm outbreak duration and study the additional impact that infection control bundle strategies (ICBSs) set up to curb coronavirus disease 2019 (COVID-19) spreading had on VREfm outbreaks. Outbreak data set were collected prospectively from April 2, 2014 to August 13, 2020 at Copenhagen University Hospital Bispebjerg, Denmark. All VREfm samples had polymerase chain reaction performed for vanA/vanB genes before whole genome sequencing using the Illumina MiSeq platform. The relatedness of isolates was studied by core genome multilocus sequence typing (cgMLST) using Ridom SeqSphere. Eighty-one outbreaks had a median outbreak duration of 32.5 days (range 5-204 days) and 1,161 VREfm isolates were sequenced. The same cgMLST cluster types reappeared after outbreaks were terminated. When comparing the first 5 months of the COVID-19 pandemic with the corresponding period in 2019, we found a 10-fold decrease in VREfm outbreak patients and median outbreak duration decreased from 56 to 7 days (88%). Several COVID-19 ICBSs were implemented from March 13 through summer 2020. VREfm outbreaks lasted up to 204 days, but our findings suggest that outbreaks might last longer since the same cgMLST persisted in the same wards for years implying an endemic situation with recurrent outbreaks caused by hospital reservoirs or readmittance of unknown VREfm carriers. The sharp decline in VREfm outbreaks during the COVID-19 pandemic was most likely due to the ICBSs, resulting in a decrease in VREfm transmission.

Evaluation of the Hologic Panther Fusion MRSA Assay for the detection of MRSA in ESwab specimens obtained from nose, throat, and perineum.

Enrichment culture (EC) remains gold standard for detecting MRSA colonisation, but molecular methods shorten turnaround time. The CE-marked automated Hologic Panther Fusion MRSA Assay (HPFM) is validated for nasal swabs. We compared HPFM with EC following an in-house PCR for detection of MRSA in nasal, pharyngeal, and perineal ESwabs. The same ESwabs were analysed using HPFM and inoculated in selective Tryptic Soy Broth (TSB) for overnight incubation. TSBs were screened by a PCR targeting nuc, femA, mecA, and mecC. Only samples with PCR results compatible with MRSA presence were inoculated onto 5% blood agar and chromogenic MRSA plates. HPFM detected MRSA in 103 of 132 EC positive samples indicating a sensitivity of 78.0% across sample types. When paired TSBs of 29 EC positive/HPFM negative samples were re-analysed by HPFM, MRSA was detected in 17/29 TSBs indicating that enrichment will increase the sensitivity of HPFM. HPFM analyses of cultured isolates from the remaining 12 EC positive/HPFM negative samples failed to detect orfX. HPFM reported the presence of MRSA in 22 samples where EC failed to identify MRSA. Fifteen of these ESwabs had been kept and direct culture without enrichment identified MRSA in seven samples. HPFM was useful for all sample sites. Compared to EC, the sensitivity of HPFM was limited because of lack of analytical sensitivity and failure to detect all MRSA variants. Failure of some MRSA-containing samples to enrich in cefoxitin-containing TSB indicates an unappreciated limitation of EC, which may lead to underestimation of the specificity of molecular assays.

Rapid Open-Source SNP-Based Clustering Offers an Alternative to Core Genome MLST for Outbreak Tracing in a Hospital Setting.

Traditional genotyping methods for infection control of antimicrobial-resistant bacteria in healthcare settings have been supplemented by whole-genome sequencing (WGS), often relying on a gene-based approach, e.g., core genome multilocus sequence typing (cgMLST), to cluster-related samples. In this study, we compared clusters of methicillin-resistant Staphylococcus aureus (MRSA) and Enterococcus faecium analyzed with the commercial cgMLST software Ridom SeqSphere+ and with an open-source single-nucleotide polymorphism (SNP)-based phylogenetic analysis pipeline (PAPABAC). A total of 5,655 MRSA and 2,572 E. faecium patient isolates, collected between 2013 and 2018, were processed. Clusters of 1,844 MRSA and 1,355 E. faecium isolates were compared to cgMLST results, and epidemiological data were included when available. The phylogenies inferred by the two different technologies were highly concordant, and the MRSA SNP tree re-captured known hospital-related outbreaks and epidemiologically linked samples. PAPABAC has the advantage over Ridom SeqSphere+ to generate stable, referable clusters without the need for sequence assembly, and it is a free-of-charge, open-source alternative to the commercial software.

Prevalence of MRSA nasal carriage among pregnant women in Copenhagen.

BACKGROUND: Methicillin resistant Staphylococcus aureus (MRSA) frequently causes outbreaks in neonatal intensive care units (NICUs). It is believed that MRSA predominantly enters the NICU with MRSA colonized parents. In Denmark, 27 MRSA NICU outbreaks have been registered between 2008 and 2019. AIM: The aim of this study was to determine the prevalence of MRSA nasal carriage in pregnant women in Copenhagen and to clarify if MRSA screening during pregnancy could add to the prevention of NICU outbreaks. METHODS: All pregnant women 18 years or older were offered MRSA nasal screening at their first midwife visit between 13 and 20 weeks of gestation. RESULTS: 1778 pregnant women were included, two (0.11%) carried MRSA in the nose. CONCLUSION: Infants of the two MRSA positive women were not admitted to a NICU and therefore the screening had no impact on NICU outbreaks. The low prevalence of MRSA found in this study does not justify MRSA screening of all pregnant women in Denmark.

Comparing whole-genome sequencing with Sanger sequencing for spa typing of methicillin-resistant Staphylococcus aureus.

spa typing of methicillin-resistant Staphylococcus aureus (MRSA) has traditionally been done by PCR amplification and Sanger sequencing of the spa repeat region. At Hvidovre Hospital, Denmark, whole-genome sequencing (WGS) of all MRSA isolates has been performed routinely since January 2013, and an in-house analysis pipeline determines the spa types. Due to national surveillance, all MRSA isolates are sent to Statens Serum Institut, where the spa type is determined by PCR and Sanger sequencing. The purpose of this study was to evaluate the reliability of the spa types obtained by 150-bp paired-end Illumina WGS. MRSA isolates from new MRSA patients in 2013 (n = 699) in the capital region of Denmark were included. We found a 97% agreement between spa types obtained by the two methods. All isolates achieved a spa type by both methods. Nineteen isolates differed in spa types by the two methods, in most cases due to the lack of 24-bp repeats in the whole-genome-sequenced isolates. These related but incorrect spa types should have no consequence in outbreak investigations, since all epidemiologically linked isolates, regardless of spa type, will be included in the single nucleotide polymorphism (SNP) analysis. This will reveal the close relatedness of the spa types. In conclusion, our data show that WGS is a reliable method to determine the spa type of MRSA.

Nasal carriage of methicillin resistant staphylococci.

Coagulase-negative staphylococci (CoNS) are believed to function as reservoirs, as well as possible sources of staphylococcal chromosome cassette mec (SCCmec) to Staphylococcus aureus, but the frequency, preferred partners, and factors promoting SCCmec transfer are not known. Such postulated in vivo genetic transfer events are likely to occur at anatomical sites such as the normal nasal mucosa, which is known to be colonized by both CoNS and coagulase positive staphylococci. In this study, we characterized S. aureus and CoNS strains colonizing the anterior nares of 67 patients in Denmark. A total of 54 patients (80%) were colonized with staphylococci that included nine different species identified by internal transcribed spacer PCR (ITS-PCR) and 16S RNA sequencing. The highest rates of colonization were found for S. epidermidis (58%) and S. aureus (39%). Methicillin resistance was present in S. aureus (53%), S. epidermidis (53%), S. haemolyticus (33%), and S. hominis (62%). Genetic backgrounds were characterized by spa typing for S. aureus and by pulsed-field gel electrophoresis for CoNS. SCCmec typing showed that SCCmec type IV (2B) was the most common in the entire collection (65%). Carriage of multiple species was detected in 20 patients (30%), 16 of whom were colonized with both S. aureus and S. epidermidis. In two cases, simultaneous carriage of different methicillin resistant species was detected. However, the strains carried different SCCmec types. Additional studies in the same epidemiological settings are warranted to identify interspecific genetic events that involve the acquisition of SCCmec by S. aureus.

Evolution of methicillin-resistant Staphylococcus aureus towards increasing resistance.

OBJECTIVES: To elucidate the evolutionary history of Staphylococcus aureus clonal complex (CC) 8, which encompasses several globally distributed epidemic lineages, including hospital-associated methicillin-resistant S. aureus (MRSA) and the highly prevalent community-associated MRSA clone USA300. METHODS: We reconstructed the phylogeny of S. aureus CC8 by mutation discovery at 112 genetic housekeeping loci from each of 174 isolates, sampled on five continents between 1957 and 2008. The distribution of antimicrobial resistance traits and of diverse mobile genetic elements was investigated in relation to the isolates' phylogeny. RESULTS: Our analyses revealed the existence of nine phylogenetic clades within CC8. We identified at least eight independent events of methicillin resistance acquisition in CC8 and dated the origin of a methicillin-resistant progenitor of the notorious USA300 clone to the mid-1970s. Of the S. aureus isolates in our collection, 88% carried plasmidic rep gene sequences, with up to five different rep genes in individual isolates and a total of eight rep families. Mapping the plasmid content onto the isolates' phylogeny illustrated the stable carriage over decades of some plasmids and the more volatile nature of others. Strikingly, we observed trends of increasing antibiotic resistance during the evolution of several lineages, including USA300. CONCLUSIONS: We propose a model for the evolution of S. aureus CC8, involving a split into at least nine phylogenetic lineages and a subsequent series of acquisitions and losses of mobile genetic elements that carry diverse virulence and antimicrobial resistance traits. The evolution of MRSA USA300 towards resistance to additional antibiotic classes is of major concern.

Structural variations of staphylococcal cassette chromosome mec type IVa in Staphylococcus aureus clonal complex 8 and unrelated lineages.

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.

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.

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.

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.

Rapid increase of genetically diverse methicillin-resistant Staphylococcus aureus, Copenhagen, Denmark.

In Copenhagen, methicillin-resistant Staphylococcus aureus (MRSA) accounted for <15 isolates per year during 1980-2002. However, since 2003 an epidemic increase has been observed, with 33 MRSA cases in 2003 and 110 in 2004. We analyzed these 143 cases epidemiologically and characterized isolates by pulsed-field gel electrophoresis, Staphylococcus protein A (spa) typing, multilocus sequence typing, staphylococcal chromosome cassette (SCC) mec typing, and detection of Panton-Valentine leukocidin (PVL) genes. Seventy-one percent of cases were community-onset MRSA (CO-MRSA); of these, 36% had no identified risk factors. We identified 29 spa types (t) and 16 sequence types (STs) belonging to 8 clonal complexes and 3 ST singletons. The most common clonal types were t024/ST8-IV, t019/ST30-IV, t044/ST80-IV, and t008/ST8-IV (USA300). A total of 86% of isolates harbored SCCmec IV, and 44% had PVL. Skin and soft tissue infections dominated. CO-MRSA with diverse genetic backgrounds is rapidly emerging in a low MRSA prevalence area.