Kit Boye

High Interlaboratory Reproducibility of DNA Sequence-Based Typing of Bacteria in a Multicenter Study

ABSTRACT Current DNA amplification-based typing methods for bacterial pathogens often lack interlaboratory reproducibility. In this international study, DNA sequence-based typing of the Staphylococcus aureus protein A gene (spa, 110 to 422 bp) showed 100% intra- and interlaboratory reproducibility without extensive harmonization of protocols for 30 blind-coded S. aureus DNA samples sent to 10 laboratories. Specialized software for automated sequence analysis ensured a common typing nomenclature.

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.

Phylogeographic variation in recombination rates within a global clone of methicillin-resistant Staphylococcus aureus

BackgroundNext-generation sequencing (NGS) is a powerful tool for understanding both patterns of descent over time and space (phylogeography) and the molecular processes underpinning genome divergence in pathogenic bacteria. Here, we describe a synthesis between these perspectives by employing a recently developed Bayesian approach, BRATNextGen, for detecting recombination on an expanded NGS dataset of the globally disseminated methicillin-resistant Staphylococcus aureus (MRSA) clone ST239.ResultsThe data confirm strong geographical clustering at continental, national and city scales and demonstrate that the rate of recombination varies significantly between phylogeographic sub-groups representing independent introductions from Europe. These differences are most striking when mobile non-core genes are included, but remain apparent even when only considering the stable core genome. The monophyletic ST239 sub-group corresponding to isolates from South America shows heightened recombination, the sub-group predominantly from Asia shows an intermediate level, and a very low level of recombination is noted in a third sub-group representing a large collection from Turkey.ConclusionsWe show that the rapid global dissemination of a single pathogenic bacterial clone results in local variation in measured recombination rates. Possible explanatory variables include the size and time since emergence of each defined sub-population (as determined by the sampling frame), variation in transmission dynamics due to host movement, and changes in the bacterial genome affecting the propensity for recombination.

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.

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.

Non-spa-Typeable Clinical Staphylococcus aureus Strains Are Naturally Occurring Protein A Mutants

ABSTRACT Staphylococcus aureus is a major human pathogen responsible for increasing the prevalence of community- and hospital-acquired infections. Protein A (SpA) is a key virulence factor of S. aureus and is highly conserved. Sequencing of the variable-number tandem-repeat region of SpA (spa typing) provides a rapid and reliable method for epidemiological studies. Rarely, non-spa-typeable S. aureus strains are encountered. The reason for this is not known. In this study, we characterized eight non-spa-typeable bacteremia isolates. Sequencing of the entire spa locus was successful for five strains and revealed various mutations of spa, all of which included a deletion of immunoglobulin G binding domain C, in which the upper primer for spa typing is located, while two strains were truly spa negative. This is the first report demonstrating that nontypeability of S. aureus by spa sequencing is due either to mutation or to a true deficiency of spa.

Changing Epidemiology of Methicillin-Resistant Staphylococcus aureus in Iceland from 2000 to 2008: a Challenge to Current Guidelines

ABSTRACT The epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) is continuously changing. Iceland has a low incidence of MRSA. A “search and destroy” policy (screening patients with defined risk factors and attempting eradication in carriers) has been implemented since 1991. Clinical and microbiological data of all MRSA patients from the years 2000 to 2008 were collected prospectively. Isolates were characterized by pulsed-field gel electrophoresis (PFGE), sequencing of the repeat region of the Staphylococcus protein A gene (spa typing), staphylococcal cassette chromosome mec (SCCmec) typing, and screening for the Panton-Valentine leukocidin (PVL) gene. Two hundred twenty-six infected (60%) or colonized (40%) individuals were detected (annual incidence 2.5 to 16/100,000). From 2000 to 2003, two health care-associated outbreaks dominated (spa types t037 and t2802), which were successfully controlled with extensive infection control measures. After 2004, an increasing number of community-associated (CA) cases without relation to the health care system occurred. A great variety of clones (40 PFGE types and 49 spa types) were found, reflecting an influx of MRSA from abroad. The USA300 and Southwest Pacific (SWP) clones were common. SCCmec type IV was most common (72%), and 38% of the isolates were PVL positive. The incidence of MRSA in Iceland has increased since 1999 but remains low and has been stable in the last years. The search and destroy policy was effective to control MRSA in the health care setting. However, MRSA in Iceland is now shifting into the community, challenging the current Icelandic guidelines, which are tailored to the health care system.

Growth phase-dependent regulation of the global virulence regulator Rot in clinical isolates of Staphylococcus aureus.

Current models for global virulence regulation in Staphylococcus aureus are mainly based on studies performed with only a limited number of laboratory strains derived from NCTC8325. In these strains the small regulatory RNA, RNAIII, has a central role in virulence gene regulation. Recently, RNAIII was suggested to control transcription of target genes partly by inhibiting translation of the transcriptional regulator Rot. The present study was undertaken to examine if the model for RNAIII/Rot-dependent virulence regulation is conserved among clinical strains. To this end, we used Rot antibodies to directly assess the amount of Rot protein in 4 well-characterized S. aureus laboratory strains (8325-4, COL, Newman, and UAMS-1) and in 9 strains of clinical origin (encompassing USA300 and Mu50). Additionally, the cellular amount of RNAIII and rot mRNA was determined in all strains. The experiments revealed considerable variation in the Rot and RNAIII levels between strains. However, in the majority of strains the cellular amount of Rot was inversely correlated to the RNAIII level. As we demonstrate that Rot is a stable protein and that the level of rot transcript appeared similar in all strains, our data support that the model for RNAIII-mediated inhibition of rot mRNA translation is conserved among clinical strains. Assessment of Rot-dependent regulation of target genes revealed that Rot is a positive regulator of spa (protein A) transcription in all strains examined. In contrast, Rot repression of sspA (serine protease) and hlb (beta-hemolysin) transcription was not conserved between strains. From this study, we conclude that while the paradigm for understanding RNAIII-dependent regulation of Rot is well-conserved, regulation of single genes is subject to considerable strain variation. We propose that variation in global regulatory networks contribute considerably to the phenotypic variation observed between S. aureus isolates.

Nosocomial transmission of community-associated methicillin-resistant Staphylococcus aureus in Danish Hospitals

OBJECTIVES The emergence of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) has changed the epidemiology of MRSA infections worldwide. In contrast to hospital-associated MRSA (HA-MRSA), CA-MRSA more frequently affects healthy individuals, both with and without recent healthcare exposure. Despite obvious epidemiological differences, it is unknown whether differences in nosocomial transmissibility exist. We have, therefore, quantified the transmissibility, expressed by the single admission reproduction number (R(A)), of CA-MRSA and HA-MRSA in hospital settings in Denmark. METHODS MRSA index cases and secondary cases were investigated in four hospitals in the Copenhagen area. Index cases were defined as non-isolated, non-screened patients with MRSA, and secondary cases were defined as persons carrying MRSA isolates-identical to that of the corresponding index-as identified through contact screening. CA-MRSA and HA-MRSA were categorized upon genotyping [CA-MRSA: t008-ST8, PVL+; t019-ST30, PVL+; t127-ST1, PVL+; t044-ST80, PVL+; and their related spa types; and HA-MRSA: all other (where ST stands for sequence type and PVL stands for Panton-Valentine leucocidin)]. A mathematical model was applied to determine the genotype-specific transmission rate (i.e. R(A)) of CA-MRSA and HA-MRSA strains. RESULTS During the 7 year study period there were 117 MRSA index cases with subsequent post-contact screening (of 1108 patients and healthcare workers), revealing 22 outbreaks with a total of 52 secondary patients. R(A) values were 0.07 (95% CI 0.00-0.28) and 0.65 (95% CI 0.48-0.84) for CA-MRSA and HA-MRSA, respectively. CONCLUSIONS In four Danish hospitals the nosocomial transmission rate of CA-MRSA was 9.3 times lower than that of HA-MRSA.

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.