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Background: Streptococcus pneumoniae is a commensal of the human upper respiratory tract and a major cause of morbidity and mortality worldwide. This paper presents the distribution of serotypes and antimicrobial resistance in commensal S. pneumoniae strains cultured from healthy carriers older than four years of age in nine European countries., Methods: Nasal swabs from healthy persons (age between 4 and 107 years old) were obtained by general practitioners from each country from November 2010 to August 2011. Swabs were cultured for S. pneumoniae using a standardized protocol. Antibiotic resistance was determined for isolated S. pneumoniae by broth microdilution. Capsular sequencing typing was used to identify serotypes, followed by serotype-specific PCR assays in case of ambiguous results., Results: Thirty-two thousand one hundred sixty-one nasal swabs were collected from which 937 S. pneumoniae were isolated. A large variation in serotype distribution and antimicrobial resistant serotypes across the participating countries was observed. Pneumococcal vaccination was associated with a higher risk of pneumococcal colonization and antimicrobial resistance independently of country and vaccine used, either conjugate vaccine or PPV 23)., Conclusions: Serotype 11A was the most common in carriage followed by serotypes 23A and 19A. The serotypes showing the highest resistance to penicillin were 14 followed by 19A. Serotype 15A showed the highest proportion of multidrug resistance.

Since 2007, livestock-associated meticillin-resistant Staphylococcus aureus (LA-MRSA) has become the predominant MRSA clade isolated from humans in the Netherlands. To assess possible temporal changes, we molecularly characterised over 9,000 LA-MRSA isolates submitted from 2003 to 2014 to the Dutch MRSA surveillance. After an initial rapid increase with a peak in 2009 (n = 1,368), the total number of submitted LA-MRSA isolates has been slowly decreasing to 968 in 2014 and over 80% of LA-MRSA belonged to one of three predominant MLVA/spa-types. Next generation sequencing (n=118) showed that MT569/t034 isolates were genetically more diverse than MT398/t011 and MT572/t108. Concurrent with the decrease in LA-MRSA, fewer people reported having contact with livestock and this was most prominent for people carrying MT569/t034 LA-MRSA. The proportion of LA-MRSA isolated from infection-related materials increased from 6% in 2009, to 13% in 2014 and most of these isolates originated from patients older than 50 years of age. Remarkably, 83% of these patients reported not having contact with livestock. The results reveal an ongoing change in the genotypic and epidemiological characteristics of Dutch LA-MRSA isolated from humans with the emergence of a LA-MRSA subclade independent of livestock exposure, suggesting LA-MRSA starts to resemble non-LA-MRSA in terms of transmissibility and pathogenicity.

To compare the genetic population structure of Staphylococcus aureus from China and Europe, 1294 human isolates were characterized by multiple-locus variable number tandem repeat analysis (MLVA). In total, MLVA identified 17 MLVA complexes (MCs), comprising 260 MLVA types (MTs) among the Chinese isolates and 372 MTs among the European isolates. The five most frequent MCs among the Chinese isolates belonged to MC398, MC5 subclade a, MC8, MC437 and MC7 and made up 55% of the sample. For the European isolates, the five most frequent MCs consisted of MC5 subclade a, MC45, MC8, MC30 and MC22, which accounted for 64% of the sample. Phylogeographic analysis of the major MCs shared between China and Europe points to a European origin of MC8 but cannot provide a consistent signal for MC5 subclade a, probably indicating a different origin. Diversity and frequency distributions of other lineages were also compared. Altogether, this study provides the first snapshot of two extant populations of S. aureus from Europe and China, and important clues on the emergence and dissemination of different lineages of S. aureus., (Copyright © 2015 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Background: Vaccination of infants with pneumococcal conjugate vaccines (PCV) has resulted in major shifts in circulating serotypes., Aim: To investigate the impact of PCV7 on the clonal composition of the pneumococcal population, and the relation of clonal lineages and clinical outcome., Materials & Methods: By using multiple-locus variable number of tandem repeat analysis, we assessed the pneumococcal populations before (n = 1154), 2-3 years after (n = 1190) and 4-6 years after (n = 1244) the introduction of PCV7 in The Netherlands., Results: We found statistically significant shifts in clonal lineages within serotypes 1 and 12F based on multiple-locus variable number of tandem repeat analysis results after the implementation of PCV7. Within serotype 12F, the increasing clonal lineage was significantly more associated with pneumonia., Conclusion: Shifts in clonal lineages within serotypes could impact the outcomes of pneumococcal disease and fill the niche of the vaccine serotypes.

A new phenotypic test, called the Carbapenem Inactivation Method (CIM), was developed to detect carbapenemase activity in Gram-negative rods within eight hours. This method showed high concordance with results obtained by PCR to detect genes coding for the carbapenemases KPC, NDM, OXA-48, VIM, IMP and OXA-23. It allows reliable detection of carbapenemase activity encoded by various genes in species of Enterobacteriaceae (e.g., Klebsiella pneumoniae, Escherichia coli and Enterobacter cloacae), but also in non-fermenters Pseudomonas aeruginosa and Acinetobacter baumannii. The CIM was shown to be a cost-effective and highly robust phenotypic screening method that can reliably detect carbapenemase activity.

Aim: Assess the best approach to type methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcal protein A (spa) typing, multiple-locus variable number tandem repeat analysis (MLVA) or both., Materials & Methods: Discriminatory power of spa typing and MLVA was determined using 20,771 MRSA isolates., Results: There were twice as many MLVA types (MTs) as spa types present in the collection. Among the top 70% of the isolates, 37 spa types and 139 MTs were found. MLVA diversity among the top-10 spa types was high (diversity index 0.96), while spa diversity among the top-10 MTs was much lower (diversity index 0.83). The probability that two MRSA isolates with the same spa type also had the same MT was low (Wallace's coefficient 0.27). By contrast, most MRSA isolates yielding the same MT also had the same spa type (Wallace's coefficient 0.90)., Conclusion: MLVA is superior to spa typing and will suffice to characterize MRSA isolates for surveillance.

Staphylococcus aureus is one of the most important human pathogens and meticillin-resistant S. aureus (MRSA) presents a major cause of healthcare- and community-acquired infections. This study investigated the spatial and temporal changes of S. aureus causing bacteraemia in Europe over a five-year interval and explored the possibility of integrating pathogen-based typing data with epidemiological and clinical information at a European level. Between January 2011 and July 2011, 350 laboratories serving 453 hospitals in 25 countries collected 3,753 isolates (meticillin-sensitive S. aureus (MSSA) and MRSA) from patients with S. aureus bloodstream infections. All isolates were sent to the national staphylococcal reference laboratories and characterised by quality-controlled spa typing. Data were uploaded to an interactive web-based mapping tool. A wide geographical distribution of spa types was found, with some prevalent in all European countries. MSSA was more diverse than MRSA. MRSA differed considerably between countries with major international clones expanding or receding when compared to a 2006 survey. We provide evidence that a network approach of decentralised typing and visualisation of aggregated data using an interactive mapping tool can provide important information on the dynamics of S. aureus populations such as early signalling of emerging strains, cross-border spread and importation by travel.

The opportunistic pathogen Staphylococcus aureus is known to interfere with wound healing and represents a significant risk factor for wound infections and invasive disease. It is generally assumed that one individual is predominantly colonized by one S. aureus type. Nevertheless, patients with the genetic blistering disease epidermolysis bullosa (EB) often carry multiple S. aureus types. We therefore investigated whether different S. aureus types are present in individual wounds of EB patients and, if so, how they are spatially distributed. The staphylococcal topography in chronic wounds was mapped by replica-plating of used bandages and subsequent typing of S. aureus isolates. Individual chronic wounds of five patients contained up to six different S. aureus types. Unexpectedly, distinct S. aureus types formed micro-colonies that were located in close proximity and sometimes even overlapped. While some adjacent S. aureus isolates were closely related, others belonged to distinct molecular complexes. We conclude that the general assumption that one individual is predominantly colonized by one type of S. aureus does not apply to chronic wounds of EB patients. We consider this observation important, not only for EB patients, but also for other patients with chronic wounds in view of the potential risk for severe staphylococcal infections.

In 2007 in The Netherlands, 30% of all human isolates of methicillin-resistant Staphylococcus aureus (MRSA) sent to the National Institute for Public Health and the Environment could not be typed by pulsed-field gel electrophoresis (non-typable (NT)-MRSA). Molecular characterisation of the NT-MRSA isolates revealed 27 different spa types and two distinct SCCmec types, type IV and V. All NT-MRSA isolates were closely related based on spa and multi-locus sequence typing and belonged to the ST398 lineage. The rapid increase of NT-MRSA (ST398) isolates over the last years shows the importance of this relatively new clonal lineage.

Background: Molecular typing of methicillin-resistant Staphylococcus aureus (MRSA) is required to study the routes and rates of transmission of this pathogen. Currently available typing techniques are either resource-intensive or have limited discriminatory ability. Multiple-locus variable number tandem repeat analysis (MLVA) may provide an alternative high throughput molecular typing tool with high epidemiological resolution., Methodology/principal Findings: A new MLVA scheme for S. aureus was validated using 1681 S. aureus isolates collected from Dutch patients and 100 isolates from pigs. MLVA using 8 tandem repeat loci was performed in 2 multiplex PCRs and the fluorescently labeled PCR products were accurately sized on an automated DNA sequencer. The assessed number of repeats was used to create MLVA profiles consisting of strings of 8 integers that were used for categorical clustering. MLVA yielded 511 types that clustered into 11 distinct MLVA complexes which appeared to coincide with MLST clonal complexes. MLVA was at least as discriminatory as PFGE and twice as discriminatory as spa-sequence typing. There was considerable congruence between MLVA, spa-sequence typing and PFGE, at the MLVA complex level with group separation values of 95.1% and 89.2%. MLVA could not discriminate between pig-related MRSA strains isolated from humans and pigs, corroborating the high degree of relationship. MLVA was also superior in the grouping of MRSA isolates previously assigned to temporal-spatial clusters with indistinguishable SpaTypes, demonstrating its enhanced epidemiological usefulness., Conclusions: The MLVA described in this study is a high throughput, relatively low cost genotyping method for S. aureus that yields discrete and unambiguous data that can be used to assign biological meaningful genotypes and complexes and can be used for interlaboratory comparisons in network accessible databases. Results suggest that MLVA offsets the disadvantages of other high discriminatory typing approaches and represents a promising tool for hospital, national and international molecular epidemiology.

Background: Sporadic cases of CA-MRSA in persons without risk-factors for MRSA carriage are increasing., Case Presentation: We report a MRSA cluster among family members of a pig-farmer, his co-workers and his pigs. Initially a young mother was seen with mastitis due to MRSA. Six months later her baby daughter was admitted to the hospital with pneumococcal otitis. After staying five days in hospital, the baby was found to be MRSA positive. At that point it was decided to look for a possible source, such as other family members and house-hold animals, including pigs on the farm, since those were reported as a possible source of MRSA earlier. Swabs were taken from the throat and nares of family members and co-workers. A veterinarian obtained swabs from the nares, throat and perineum of 10 pigs. Swabs were cultured following a national protocol to detect MRSA that included the use of an enrichment broth. Animal and human strains were characterized by PFGE, spa-typing, MLST analysis, SSCmec, AGR typing, and the detection for PVL, LukM, and TSST toxin genes. Three family members, three co-workers, and 8 of the 10 pigs were MRSA positive. With the exception of the initial case (the mother) all persons were solely colonized, with no signs of clinical infections. After digestion with SmaI, none of the strains showed any bands using PFGE. All isolates belonged to spa type t108 and ST398., Conclusion: 1. This report clearly shows clonal spread and transmission between humans and pigs in the Netherlands. 2. MLST sequence type 398 might be of international importance as pig-MRSA, since this type was shown earlier to be present in epidemiologically unrelated French pigs and pig-farmers. 3. Research is needed to evaluate whether this is a local problem or a new source of MRSA, that puts the until now successful Search and Destroy policy of the Netherlands at risk.

The worldwide emergence of community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) can have severe public health implications. Familial transmissions of CA-MRSA in The Netherlands were investigated. Among the families studied, two clusters of CA-MRSA could be identified. This report demonstrates that family members can serve as reservoirs of CA-MRSA which may become a serious problem in containing the spread of MRSA.

Methicillin-resistant Staphylococcus aureus (MRSA) strains carrying the Panton-Valentine leucocidin (PVL) genes have been reported worldwide and are a serious threat to public health. The PVL genes encode a highly potent toxin which is involved in severe skin infections and necrotizing pneumonia, even in previously healthy individuals. We assessed the prevalence of PVL-positive MRSA in The Netherlands for two periods of time: (i) 1987 through 1995 and (ii) 2000 and 2002, and determined their characteristics by using multilocus sequence typing and staphylococcal chromosome cassette (SCCmec) typing. It was found that up to 15% of all MRSA isolates detected in The Netherlands harbored the PVL genes. Most PVL-positive MRSA isolates were obtained from severe soft tissue infections in relatively young individuals. The first PVL-positive MRSA described in The Netherlands, isolated in 1988, was a single-locus variant of the "Berlin" epidemic MRSA clone. The 20 PVL-positive MRSA isolates studied in 2000 and 2002 consisted of five different sequence types (STs) that belonged to four clonal complexes. One of the STs, ST80, is considered to be a widespread European clone and was the most predominant ST (60%) in this study, while ST37 had never been found to be associated with PVL-positive MRSA. Most isolates harbored SCCmec type IV, a supposed marker for community-acquired MRSA. The number and type of virulence-associated genes varied among the different STs.

Analysis of methicillin-resistant Staphylococcus aureus (MRSA) isolates in the Netherlands in 2003 revealed that 8% of the hospital isolates carried the loci for Panton-Valentine leukocidin (PVL). Molecular subtyping showed that most Dutch PVL-MRSA genotypes corresponded to well-documented global epidemic types. The most common PVL-MRSA genotypes were sequence type ST8, ST22, ST30, ST59 and ST80. MRSA with ST8 increased in the Netherlands from 1% in 2002 to 17% in 2003. It is emphasised that PVL-MRSA might not only emerge in the community, but also in the hospital environment.

Methicillin-resistant Staphylococcus aureus (MRSA) is an important human pathogen and represents a growing public health burden due to the emergence and spread of epidemic strains, particularly within the hospital environment. An epidemic MRSA clone, with characteristic low-level resistance to oxacillin, emerged in the year 2000 and became endemic in the Netherlands. Multilocus sequence typing characterized the strain as sequence type 45, which was previously designated the Berlin epidemic MRSA clone. In 2 years, this strain has become the predominant MRSA clone in the Netherlands.