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1.  Multilocus Sequence Typing and Further Genetic Characterization of the Enigmatic Pathogen, Staphylococcus hominis 
PLoS ONE  2013;8(6):e66496.
Staphylococcus hominis is a commensal resident of human skin and an opportunistic pathogen. The species is subdivided into two subspecies, S. hominis subsp. hominis and S. hominis subsp. novobiosepticus, which are difficult to distinguish. To investigate the evolution and epidemiology of S. hominis, a total of 108 isolates collected from 10 countries over 40 years were characterized by classical phenotypic methods and genetic methods. One nonsynonymous mutation in gyrB, scored with a novel SNP typing assay, had a perfect association with the novobiocin-resistant phenotype. A multilocus sequence typing (MLST) scheme was developed from six housekeeping gene fragments, and revealed relatively high levels of genetic diversity and a significant impact of recombination on S. hominis population structure. Among the 40 sequence types (STs) identified by MLST, three STs (ST2, ST16 and ST23) were S. hominis subsp. novobiosepticus, and they distinguished between isolates from different outbreaks, whereas 37 other STs were S. hominis subsp. hominis, one of which was widely disseminated (ST1). A modified PCR assay was developed to detect the presence of ccrAB4 from the SCCmec genetic element. S. hominis subsp. novobiosepticus isolates were oxacillin-resistant and carriers of specific components of SCCmec (mecA class A, ccrAB3, ccrAB4, ccrC), whereas S. hominis subsp. hominis included both oxacillin-sensitive and -resistant isolates and a more diverse array of SCCmec components. Surprisingly, phylogenetic analyses indicated that S. hominis subsp. novobiosepticus may be a polyphyletic and, hence, artificial taxon. In summary, these results revealed the genetic diversity of S. hominis, the identities of outbreak-causing clones, and the evolutionary relationships between subspecies and clones. The pathogenic lifestyle attributed to S. hominis subsp. novobiosepticus may have originated on more than one occasion.
doi:10.1371/journal.pone.0066496
PMCID: PMC3679023  PMID: 23776678
2.  Staphylococcus epidermidis Colonization Is Highly Clonal Across US Cardiac Centers 
The Journal of Infectious Diseases  2012;205(9):1391-1398.
Background. Little is known about the clonality of Staphylococcus epidermidis in the United States, although it is the predominant pathogen in infections involving prosthetic materials, including ventricular assist devices (VADs).
Methods. Seventy-five VAD recipients at 4 geographically diverse US cardiac centers were prospectively followed up to 1 year of VAD support. The anterior nares, sternum, and (future) driveline exit site were cultured for S. epidermidis before VAD insertion and at 7 times after surgery. Infection isolates were also collected. Isolates were typed by pulsed-field gel electrophoresis. A subset underwent susceptibility testing and staphylococcal chromosomal cassette mec and multilocus sequence typing.
Results. A total of 1559 cultures yielded 565 S. epidermidis isolates; 254 of 548 typed isolates (46%) belonged to 1 of 7 clonal types as defined by pulsed-field gel electrophoresis. These clones were identified in up to 27 people distributed across all 4 cardiac centers. They caused 3 of 6 VAD-related infections. Disseminated clones were more antibiotic resistant than were less prevalent isolates (eg, 79% vs 54% methicillin resistant; P = .0021).
Conclusions. This study revealed that healthcare–associated S. epidermidis infection is remarkably clonal. We describe S. epidermidis clones that are highly resistant to antibiotics distributed across US cardiac centers. These clones may have determinants that enhance transmissibility, persistence, or invasiveness.
Clinical Trials Registration. NCT01471795.
doi:10.1093/infdis/jis218
PMCID: PMC3324403  PMID: 22457291
3.  Extensive Dissemination of Methicillin-Resistant Staphylococcus aureus (MRSA) between the Hospital and the Community in a Country with a High Prevalence of Nosocomial MRSA 
PLoS ONE  2013;8(4):e59960.
According to the EARS-Net surveillance data, Portugal has the highest prevalence of nosocomial methicillin-resistant Staphylococcus aureus (MRSA) in Europe, but the information on MRSA in the community is very scarce and the links between the hospital and community are not known. In this study we aimed to understand the events associated to the recent sharp increase in MRSA frequency in Portugal and to evaluate how this has shaped MRSA epidemiology in the community. With this purpose, 180 nosocomial MRSA isolates recovered from infection in two time periods and 14 MRSA isolates recovered from 89 samples of skin and soft tissue infections (SSTI) were analyzed by pulsed-field gel electrophoresis (PFGE), staphylococcal chromosome cassette mec (SCCmec) typing, spa typing and multilocus sequence typing (MLST). All isolates were also screened for the presence of Panton Valentine leukocidin (PVL) and arginine catabolic mobile element (ACME) by PCR. The results showed that ST22-IVh, accounting for 72% of the nosocomial isolates, was the major clone circulating in the hospital in 2010, having replaced two previous dominant clones in 1993, the Iberian (ST247-I) and Portuguese (ST239-III variant) clones. Moreover in 2010, three clones belonging to CC5 (ST105-II, ST125-IVc and ST5-IVc) accounted for 20% of the isolates and may represent the beginning of new waves of MRSA in this hospital. Interestingly, more than half of the MRSA isolates (8/14) causing SSTI in people attending healthcare centers in Portugal belonged to the most predominant clones found in the hospital, namely ST22-IVh (n = 4), ST5-IVc (n = 2) and ST105-II (n = 1). Other clones found included ST5-V (n = 6) and ST8-VI (n = 1). None of the MRSA isolates carried PVL and only five isolates (ST5-V-t179) carried ACME type II. The emergence and spread of EMRSA-15 may be associated to the observed increase in MRSA frequency in the hospital and the consequent spillover of MRSA into the community.
doi:10.1371/journal.pone.0059960
PMCID: PMC3617237  PMID: 23593155
4.  Community-Associated Methicillin-Resistant Staphylococcus aureus Lacking PVL, as a Cause of Severe Invasive Infection Treated with Linezolid 
Case Reports in Pediatrics  2013;2013:727824.
Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) is an emerging public health problem worldwide. Severe invasive infections have been described, mostly associated with the presence of Panton-Valentine leukocidin (PVL). In Portugal limited information exists regarding CA-MRSA infections. In this study we describe the case of a previously healthy 12-year-old female, sport athlete, who presented to the hospital with acetabulofemoral septic arthritis, myositis, fasciitis, acetabulum osteomyelitis, and pneumonia. The MRSA isolated from blood and synovial fluid was PVL negative and staphylococcal enterotoxin type P (SEP) and type L (SEL) positive, with a vancomycin MIC of 1.0 mg/L and resistant to clindamycin and ciprofloxacin. The patient was submitted to multiple surgical drainages and started on vancomycin, rifampicin, and gentamycin. Due to persistence of fever and no microbiological clearance, linezolid was started with improvement. This is one of the few reported cases of severe invasive infection caused by CA-MRSA in Portugal, which was successfully treated with linezolid. In spite of the severity of infection, the MRSA isolate did not produce PVL.
doi:10.1155/2013/727824
PMCID: PMC3590555  PMID: 23509655
5.  High Genetic Diversity among Community-Associated Staphylococcus aureus in Europe: Results from a Multicenter Study 
PLoS ONE  2012;7(4):e34768.
Background
Several studies have addressed the epidemiology of community-associated Staphylococcus aureus (CA-SA) in Europe; nonetheless, a comprehensive perspective remains unclear. In this study, we aimed to describe the population structure of CA-SA and to shed light on the origin of methicillin-resistant S. aureus (MRSA) in this continent.
Methods and Findings
A total of 568 colonization and infection isolates, comprising both MRSA and methicillin-susceptible S. aureus (MSSA), were recovered in 16 European countries, from community and community-onset infections. The genetic background of isolates was characterized by molecular typing techniques (spa typing, pulsed-field gel electrophoresis and multilocus sequence typing) and the presence of PVL and ACME was tested by PCR. MRSA were further characterized by SCCmec typing. We found that 59% of all isolates were associated with community-associated clones. Most MRSA were related with USA300 (ST8-IVa and variants) (40%), followed by the European clone (ST80-IVc and derivatives) (28%) and the Taiwan clone (ST59-IVa and related clonal types) (15%). A total of 83% of MRSA carried Panton-Valentine leukocidin (PVL) and 14% carried the arginine catabolic mobile element (ACME). Surprisingly, we found a high genetic diversity among MRSA clonal types (ST-SCCmec), Simpson’s index of diversity = 0.852 (0.788–0.916). Specifically, about half of the isolates carried novel associations between genetic background and SCCmec. Analysis by BURP showed that some CA-MSSA and CA-MRSA isolates were highly related, suggesting a probable local acquisition/loss of SCCmec.
Conclusions
Our results imply that CA-MRSA origin, epidemiology and population structure in Europe is very dissimilar from that of USA.
doi:10.1371/journal.pone.0034768
PMCID: PMC3338755  PMID: 22558099
6.  S3QL: A distributed domain specific language for controlled semantic integration of life sciences data 
BMC Bioinformatics  2011;12:285.
Background
The value and usefulness of data increases when it is explicitly interlinked with related data. This is the core principle of Linked Data. For life sciences researchers, harnessing the power of Linked Data to improve biological discovery is still challenged by a need to keep pace with rapidly evolving domains and requirements for collaboration and control as well as with the reference semantic web ontologies and standards. Knowledge organization systems (KOSs) can provide an abstraction for publishing biological discoveries as Linked Data without complicating transactions with contextual minutia such as provenance and access control.
We have previously described the Simple Sloppy Semantic Database (S3DB) as an efficient model for creating knowledge organization systems using Linked Data best practices with explicit distinction between domain and instantiation and support for a permission control mechanism that automatically migrates between the two. In this report we present a domain specific language, the S3DB query language (S3QL), to operate on its underlying core model and facilitate management of Linked Data.
Results
Reflecting the data driven nature of our approach, S3QL has been implemented as an application programming interface for S3DB systems hosting biomedical data, and its syntax was subsequently generalized beyond the S3DB core model. This achievement is illustrated with the assembly of an S3QL query to manage entities from the Simple Knowledge Organization System. The illustrative use cases include gastrointestinal clinical trials, genomic characterization of cancer by The Cancer Genome Atlas (TCGA) and molecular epidemiology of infectious diseases.
Conclusions
S3QL was found to provide a convenient mechanism to represent context for interoperation between public and private datasets hosted at biomedical research institutions and linked data formalisms.
doi:10.1186/1471-2105-12-285
PMCID: PMC3155508  PMID: 21756325
S3DB; Linked Data; KOS; RDF; SPARQL; knowledge organization system, policy
7.  Molecular Epidemiology of Methicillin-Resistant Staphylococcus hominis (MRSHo): Low Clonality and Reservoirs of SCCmec Structural Elements 
PLoS ONE  2011;6(7):e21940.
Background
Methicillin resistant Staphylococcus hominis (MRSHo) are important human pathogens in immunocompromised patients. However, little is known regarding its population structure and staphylococcal chromosomal cassette mec (SCCmec) content.
Methodology/Principal Findings
To assess the population structure and the SCCmec content of S. hominis, 34 MRSHo and 11 methicillin-susceptible S. hominis (MSSHo) from neutropenic patients collected over a 3-year period were studied. The genetic backgrounds of S. hominis isolates were analyzed by pulsed-field gel electrophoresis (PFGE) and SCCmec types were determined by PCR. Cassette chromosome recombinases (ccr) were characterized by PCR and ccrB sequencing. The 34 S. hominis isolates were classified into as many as 28 types and 32 subtypes (SID = 99.82%); clonal dissemination was occasionally observed. The main SCCmec structures identified were SCCmec type VI (4B) (20%), SCCmec VIII (4A) (15%), and a new SCCmec composed of mec complex A in association with ccrAB1 (38%); 27% of the isolates harbored non-typeable SCCmec. Overall, a high prevalence of mec complex A (73.5%), ccrAB1 (50%) and ccrAB4 (44%) were found. Importantly, ccrB1 and ccrB4 from both MRSHo and MSSHo showed a high nucleotide sequence homology with those found in S. aureus SCCmec I, VI and VIII respectively (>95%).
Conclusions/Significance
The S. hominis population showed a limited clonality and a low genetic diversity in the allotypes of ccr and classes of mec complex. Moreover, our data suggest that S. hominis might have been a privileged source of mec complex A, ccrB1 and ccrB4, for the assembly of primordial SCCmec types.
doi:10.1371/journal.pone.0021940
PMCID: PMC3132775  PMID: 21760926
8.  Large screening of CA-MRSA among Staphylococcus aureus colonizing healthy young children living in two areas (urban and rural) of Portugal 
BMC Infectious Diseases  2010;10:110.
Background
The incidence of pediatric infections due to community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA), including children with no identifiable risk factors, has increased worldwide in the last decade. This suggests that healthy children may constitute a reservoir of MRSA in the community. In this study, nested within a larger one on nasopharyngeal ecology, we aimed to: (i) evaluate the prevalence of MRSA colonizing young children in Portugal; and (ii) compare results with those obtained in a study conducted a decade ago, when this prevalence was <0.5%.
Methods
In the years 2006, 2007, and 2009, nasopharyngeal samples were obtained from 2,100 children aged up to 6 years attending day-care centers. S. aureus were isolated by routine procedures and strains were tested for susceptibility against a panel of 12 antimicrobial agents. MRSA isolates were further characterized by SmaI-PFGE profiling, MLST, spa typing, SCCmec typing, and presence of virulence factors.
Results
Seventeen percent of the children carried S. aureus. Among the 365 isolates, non-susceptibility rates were 88% to penicillin, 14% to erythromycin, 6% to clindamycin, 2% to tetracycline, and <1% to oxacillin, rifampicin, ciprofloxacin, and SXT. Three MRSA strains were isolated. These had properties of CA-MRSA, such as low-level resistance to oxacillin and limited resistance to non-beta-lactams. Two CA-MRSA were related to USA700 (ST72-IV): one was ST72-IVc, spa type t148; the other was ST939-IVa (ST939 is a single locus variant (SLV) of ST72), spa type t324. The third strain was related to USA300 (ST8-IV) being characterized by ST931 (SLV of ST8)-VI, spa type t008. The three MRSA strains were PVL-negative, but all carried LukE-LukD leukocidin, hemolysins gamma, gamma variant and beta, and staphylococcal enterotoxin sel.
Conclusions
Our results, based on analysis of S. aureus isolated from nasopharyngeal samples, suggest that in Portugal the prevalence of CA-MRSA carriage in healthy young children remains extremely low favoring the exclusion of this group as a reservoir of such isolates.
doi:10.1186/1471-2334-10-110
PMCID: PMC2876167  PMID: 20438633
9.  Genetic Diversity of Arginine Catabolic Mobile Element in Staphylococcus epidermidis 
PLoS ONE  2009;4(11):e7722.
Background
The methicillin-resistant Staphylococcus aureus clone USA300 contains a novel mobile genetic element, arginine catabolic mobile element (ACME), that contributes to its enhanced capacity to grow and survive within the host. Although ACME appears to have been transferred into USA300 from S. epidermidis, the genetic diversity of ACME in the latter species remains poorly characterized.
Methodology/Principal Findings
To assess the prevalence and genetic diversity of ACME, 127 geographically diverse S. epidermidis isolates representing 86 different multilocus sequence types (STs) were characterized. ACME was found in 51% (65/127) of S. epidermidis isolates. The vast majority (57/65) of ACME-containing isolates belonged to the predominant S. epidermidis clonal complex CC2. ACME was often found in association with different allotypes of staphylococcal chromosome cassette mec (SCCmec) which also encodes the recombinase function that facilities mobilization ACME from the S. epidermidis chromosome. Restriction fragment length polymorphism, PCR scanning and DNA sequencing allowed for identification of 39 distinct ACME genetic variants that differ from one another in gene content, thereby revealing a hitherto uncharacterized genetic diversity within ACME. All but one ACME variants were represented by a single S. epidermidis isolate; the singular variant, termed ACME-I.02, was found in 27 isolates, all of which belonged to the CC2 lineage. An evolutionary model constructed based on the eBURST algorithm revealed that ACME-I.02 was acquired at least on 15 different occasions by strains belonging to the CC2 lineage.
Conclusions/Significance
ACME-I.02 in diverse S. epidermidis isolates were nearly identical in sequence to the prototypical ACME found in USA300 MRSA clone, providing further evidence for the interspecies transfer of ACME from S. epidermidis into USA300.
doi:10.1371/journal.pone.0007722
PMCID: PMC2768820  PMID: 19893740
10.  Improved Multilocus Sequence Typing Scheme for Staphylococcus epidermidis▿  
Journal of Clinical Microbiology  2006;45(2):616-619.
We evaluated three multilocus sequence typing (MLST) schemes for Staphylococcus epidermidis and selected the seven most discriminatory loci for the formation of a new, more powerful MLST scheme. This improved scheme gave 31 sequence types (STs) and 5 clonal complexes (CCs), whereas the other schemes delineate 16 to 24 STs and 1 to 3 CCs.
doi:10.1128/JCM.01934-06
PMCID: PMC1829011  PMID: 17151213
11.  Molecular Characterization of Methicillin-Resistant Staphylococcus epidermidis Clones: Evidence of Geographic Dissemination 
Journal of Clinical Microbiology  2002;40(2):430-438.
Denmark and Iceland are countries where the frequency of methicillin-resistant Staphylococcus aureus is very low due to strict infection control and restrictive antibiotic use policies. In contrast, methicillin-resistant S. epidermidis (MRSE) continues to be isolated as a nosocomial pathogen. The molecular typing by pulsed-field gel electrophoresis (PFGE) of 136 MRSE isolates from five hospitals in Denmark and 94 MRSE isolates from one hospital in Iceland collected in 1997 and 1998 defined 40 different patterns. Closely related PFGE types were found in isolates recovered in Iceland, Denmark, Mexico, Uruguay, Greece, and Cape Verde, evidencing for the first time the geographic clonal dissemination of MRSE strains. The large majority (87.4%) of the MRSE isolates studied were multiresistant.
doi:10.1128/JCM.40.2.430-438.2002
PMCID: PMC153385  PMID: 11825953
12.  Identification of Clinical Staphylococcal Isolates from Humans by Internal Transcribed Spacer PCR 
Journal of Clinical Microbiology  2001;39(9):3099-3103.
The emergence of coagulase-negative staphylococci not only as human pathogens but also as reservoirs of antibiotic resistance determinants requires the deployment and development of methods for their rapid and reliable identification. Internal transcribed spacer-PCR (ITS-PCR) was used to identify a collection of 617 clinical staphylococcal isolates. The amplicons were resolved in high-resolution agarose gels and visually compared with the patterns obtained for the control strains of 29 staphylococcal species. Of the 617 isolates studied, 592 (95.95%) were identified by ITS-PCR and included 11 species: 302 isolates of Staphylococcus epidermidis, 157 of S. haemolyticus, 79 of S. aureus, 21 of S. hominis, 14 of S. saprophyticus, 8 of S. warneri, 6 of S. simulans, 2 of S. lugdunensis, and 1 each of S. caprae, S. carnosus, and S. cohnii. All species analyzed had unique ITS-PCR patterns, although some were very similar, namely, the group S. saprophyticus, S. cohnii, S. gallinarum, S. xylosus, S. lentus, S. equorum, and S. chromogenes, the pair S. schleiferi and S. vitulus, and the pair S. piscifermentans and S. carnosus. Four species, S. aureus, S. caprae, S. haemolyticus, and S. lugdunensis, showed polymorphisms on their ITS-PCR patterns. ITS-PCR proved to be a valuable alternative for the identification of staphylococci, offering, within the same response time and at lower cost, higher reliability than the currently available commercial systems.
doi:10.1128/JCM.39.9.3099-3103.2001
PMCID: PMC88303  PMID: 11526135
13.  Three-Year Assessment of Methicillin-Resistant Staphylococcus aureus Clones in Latin America from 1996 to 1998 
Journal of Clinical Microbiology  2001;39(6):2197-2205.
Four hundred ninety-nine methicillin-resistant Staphylococcus aureus (MRSA) isolates recovered from 1996 to 1998 from 22 hospitals in five countries of Latin America—Argentina, Brazil, Chile, Uruguay and Mexico—were examined for antimicrobial susceptibility and clonal type in order to define the endemic clones in those hospitals. The hybridization of ClaI restriction digests with the mecA- and Tn554-specific DNA probes combined with pulsed-field gel electrophoresis of chromosomal SmaI digests (ClaI-mecA::ClaI-Tn554::PFGE clonal types) documented not only the predominance and persistence of the Brazilian clone (XI::B::B) in Brazil (97%) and Argentina (86%) but also its massive dissemination to Uruguay (100%). Moreover, a close relative of the Brazilian clone (XI::κ::B) was highly represented in Chile (53%) together with a novel clone (47%) (II::E′::F) resistant to pencillin, oxacillin, ciprofloxacin, chloramphenicol, clindamycin, erythromycin, and gentamicin. A unique clonal type (I::NH::M) was detected in Mexico among pediatric isolates and was resistant to penicillin, oxacillin, and gentamicin only. This study clearly documented the very large capacity for geographic expansion and the persistence of the Brazilian clone, contributing not only to the increasing uniformity of the MRSA in South America but worldwide as well.
doi:10.1128/JCM.39.6.2197-2205.2001
PMCID: PMC88111  PMID: 11376057

Results 1-13 (13)