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1.  Non-typeable pneumococci circulating in Portugal are of cps type NCC2 and have genomic features typical of encapsulated isolates 
BMC Genomics  2014;15(1):863.
Background
Pneumococcus is a major human pathogen and the polysaccharide capsule is considered its main virulence factor. Nevertheless, strains lacking a capsule, named non-typeable pneumococcus (NT), are maintained in nature and frequently colonise the human nasopharynx. Interest in these strains, not targeted by any of the currently available pneumococcal vaccines, has been rising as they seem to play an important role in the evolution of the species. Currently, there is a paucity of data regarding this group of pneumococci. Also, questions have been raised on whether they are true pneumococci. We aimed to obtain insights in the genetic content of NT and the mechanisms leading to non-typeability and to genetic diversity.
Results
A collection of 52 NT isolates representative of the lineages circulating in Portugal between 1997 and 2007, as determined by pulsed-field gel electrophoresis and multilocus sequence typing, was analysed. The capsular region was sequenced and comparative genomic hybridisation (CGH) using a microarray covering the genome of 10 pneumococcal strains was carried out. The presence of mobile elements was investigated as source of intraclonal variation. NT circulating in Portugal were found to have similar capsular regions, of cps type NCC2, i.e., having aliB-like ORF1 and aliB-like ORF2 genes. The core genome of NT was essentially similar to that of encapsulated strains. Also, competence genes and most virulence genes were present. The few virulence genes absent in all NT were the capsular genes, type-I and type-II pili, choline-binding protein A (cbpA/pspC), and pneumococcal surface protein A (pspA). Intraclonal variation could not be entirely explained by the presence of prophages and other mobile elements.
Conclusions
NT circulating in Portugal are a homogeneous group belonging to cps type NCC2. Our observations support the theory that they are bona-fide pneumococcal isolates that do not express the capsule but are otherwise essentially similar to encapsulated pneumococci. Thus we propose that NT should be routinely identified and reported in surveillance studies.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-863) contains supplementary material, which is available to authorized users.
doi:10.1186/1471-2164-15-863
PMCID: PMC4200197  PMID: 25283442
2.  Insights into Alpha-Hemolysin (Hla) Evolution and Expression among Staphylococcus aureus Clones with Hospital and Community Origin 
PLoS ONE  2014;9(7):e98634.
Background
Alpha-hemolysin (Hla) is a major virulence factor in the pathogenesis of Staphylococcus aureus infection, being active against a wide range of host cells. Although hla is ubiquitous in S. aureus, its genetic diversity and variation in expression in different genetic backgrounds is not known. We evaluated nucleotide sequence variation and gene expression profiles of hla among representatives of hospital (HA) and community-associated (CA) S. aureus clones.
Methods
51 methicillin-resistant S. aureus and 22 methicillin-susceptible S. aureus were characterized by PFGE, spa typing, MLST and SCCmec typing. The internal regions of hla and the hla promoter were sequenced and gene expression was assessed by RT-PCR.
Results
Alpha-hemolysin encoding- and promoter sequences were diverse, with 12 and 23 different alleles, respectively. Based on phylogenetic analysis, we suggest that hla may have evolved together with the S. aureus genetic background, except for ST22, ST121, ST59 and ST93. Conversely, the promoter region showed lack of co-evolution with the genetic backgrounds. Four non-synonymous amino acid changes were identified close to important regions of hla activity. Amino acid changes in the RNAIII binding site were not associated to hla expression. Although expression rates of hla were in general strain-specific, we observed CA clones showed significantly higher hla expression (p = 0.003) when compared with HA clones.
Conclusion
We propose that the hla gene has evolved together with the genetic background. Overall, CA genetic backgrounds showed higher levels of hla expression than HA, and a high strain-to-strain variation of gene expression was detected in closely related strains.
doi:10.1371/journal.pone.0098634
PMCID: PMC4102472  PMID: 25033196
3.  Novel Determinants of Antibiotic Resistance: Identification of Mutated Loci in Highly Methicillin-Resistant Subpopulations of Methicillin-Resistant Staphylococcus aureus 
mBio  2014;5(2):e01000-13.
ABSTRACT
We identified mutated genes in highly resistant subpopulations of methicillin-resistant Staphylococcus aureus (MRSA) that are most likely responsible for the historic failure of the β-lactam family of antibiotics as therapeutic agents against these important pathogens. Such subpopulations are produced during growth of most clinical MRSA strains, including the four historically early MRSA isolates studied here. Chromosomal DNA was prepared from the highly resistant cells along with DNA from the majority of cells (poorly resistant cells) followed by full genome sequencing. In the highly resistant cells, mutations were identified in 3 intergenic sequences and 27 genes representing a wide range of functional categories. A common feature of these mutations appears to be their capacity to induce high-level β-lactam resistance and increased amounts of the resistance protein PBP2A in the bacteria. The observations fit a recently described model in which the ultimate controlling factor of the phenotypic expression of β-lactam resistance in MRSA is a RelA-mediated stringent response.
IMPORTANCE
It has been well established that the level of antibiotic resistance (i.e., minimum concentration of a β-lactam antibiotic needed to inhibit growth) of a methicillin-resistant Staphylococcus aureus (MRSA) strain depends on the transcription and translation of the resistance protein PBP2A. Here we describe mutated loci in an additional novel set of genetic determinants that appear to be essential for the unusually high resistance levels typical of subpopulations of staphylococci that are produced with unique low frequency in most MRSA clinical isolates. We propose that mutations in these determinants can trigger induction of the stringent stress response which was recently shown to cause increased transcription/translation of the resistance protein PBP2A in parallel with the increased level of resistance.
doi:10.1128/mBio.01000-13
PMCID: PMC3993859  PMID: 24713324
4.  The glucosaminidase domain of Atl – the major Staphylococcus aureus autolysin – has DNA-binding activity 
MicrobiologyOpen  2014;3(2):247-256.
In this communication, we describe evidence demonstrating the capacity of Atl, the major Staphylococcus aureus autolytic enzyme to bind DNA. Electrophoretic mobility shift assays (EMSA) show that both the Atl protein and the endo-β-N-acetylglucosaminidase (GL) domain were able to bind DNA of nonspecific sequence. The implications of this unexpected observation for the physiology of S. aureus remain to be explored.
doi:10.1002/mbo3.165
PMCID: PMC3996572  PMID: 24585695
Atl autolysin; DNA binding; protein-DNA interaction; Staphylococcus aureus
5.  Low Prevalence of Pneumococcal Carriage and High Serotype and Genotype Diversity among Adults over 60 Years of Age Living in Portugal 
PLoS ONE  2014;9(3):e90974.
Pneumococcal disease is frequent at the extremes of age. While several studies have looked at colonization among young children, much less is known among the elderly. We aimed to evaluate pneumococcal carriage among elderly adults living in Portugal. Between April 2010 and December 2012, nasopharyngeal and oropharyngeal swabs of adults over 60 years of age, living in an urban area (n = 1,945) or in a rural area (n = 1,416), were obtained. Pneumococci were isolated by culture-based standard procedures, identified by optochin susceptibility, bile solubility and PCR screening for lytA and cpsA, and characterized by antibiotype, serotype, and MLST. Associations between pneumococcal carriage, socio-demographic and clinical characteristics were evaluated by univariate analysis and multiple logistic regression. The global prevalence of carriage was 2.3% (95% CI: 1.8–2.8). In the multiple logistic regression analysis, smoking, being at a retirement home, and living in a rural area increased the odds of being a pneumococcal carrier by 4.4-fold (95% CI: 1.9–9.2), 2.0-fold (95% CI: 1.1–3.6) and 2.0-fold (95% CI: 1.2–3.5), respectively. Among the 77 pneumococcal isolates, 26 serotypes and 40 STs were identified. The most prevalent serotypes were (in decreasing order) 19A, 6C, 22F, 23A, 35F, 11A, and 23B, which accounted, in total, for 60.0% of the isolates. Most isolates (93.5%) had STs previously described in the MLST database. Resistance to macrolides, non-susceptibility to penicillin and multidrug resistance were found in 19.5%, 11.7%, and 15.6% of the isolates, respectively. We conclude that the prevalence of pneumococcal carriage in the elderly, in Portugal, as determined by culture-based methods, is low. Serotype and genotype diversity is high. Living in a rural area, in a retirement home, and being a smoker increased the risk of pneumococcal carriage. This study contributes to the establishment of a baseline that may be used to monitor how novel pneumococcal vaccines impact on colonization among the elderly.
doi:10.1371/journal.pone.0090974
PMCID: PMC3946249  PMID: 24604030
6.  Contamination of Public Buses with MRSA in Lisbon, Portugal: A Possible Transmission Route of Major MRSA Clones within the Community 
PLoS ONE  2013;8(11):e77812.
In a previous study we have shown that public buses in Oporto, the second largest city in Portugal, were highly contaminated with MRSA. Here we describe the results of a similar study performed in another urban area of Portugal–Lisbon, the capital. Between May 2011 and May 2012, hand touched surfaces of 199 public buses in Lisbon were screened for MRSA contamination. Subsequently, the hands of 575 passengers who frequently use these bus lines were also screened. All hand carriers of MRSA were further screened for nasal carriage. The isolates were characterized by PFGE, staphylococcal cassette chromosome (SCC) mec typing, spa typing, MLST and were tested for the presence of mecA, Panton-Valentine leukocidin and arginine catabolic mobile element genes. MRSA contamination was shown in 72 buses (36.2%). The majority of the isolates belonged to three major clones: Clone A was identified as EMRSA-15 defined by pattern PFGE A, spa types t2357/t747/t025/t379/t910, ST22, and SCCmec IVh (n = 21; 29%). Clone B was the New York/Japan clone characterized by PFGE B-t002/t10682-ST5-II (n = 15; 21%). Clone C included isolates with characteristics of the international community-acquired USA300 or related clones, PFGE C-t008-ST8-IVa/IVc/IVg/IVnt/VI (n = 19; 26%). The first two clones are currently the two major lineages circulating in Portuguese hospitals. The hands of 15 individuals were contaminated with MRSA belonging to the nosocomial clones A or B. Eleven of these individuals were not nasal carriers of MRSA and all but one had travelled by public transportation, namely by bus, prior to sampling. In conclusion, public buses in two major cities in Portugal are often contaminated with MRSA representing clones dominant in hospitals in the particular geographic area. MRSA contamination of public transport and the transfer of the bacteria to the hands of passengers may represent a route through which hospital-acquired MRSA clones may spread to the community.
doi:10.1371/journal.pone.0077812
PMCID: PMC3819345  PMID: 24223124
7.  Virulence Potential and Genome-Wide Characterization of Drug Resistant Streptococcus pneumoniae Clones Selected In Vivo by the 7-Valent Pneumococcal Conjugate Vaccine 
PLoS ONE  2013;8(9):e74867.
We used mouse models of pneumococcal colonization and disease combined with full genome sequencing to characterize three major drug resistant clones of S. pneumoniae that were recovered from the nasopharynx of PCV7-immunized children in Portugal. The three clones – serotype 6A (ST2191), serotype 15A (ST63) and serotype 19A (ST276) carried some of the same drug resistance determinants already identified in nasopharyngeal isolates from the pre-PCV7 era. The three clones were able to colonize efficiently the mouse nasopharyngeal mucosa where populations of these pneumococci were retained for as long as 21 days. During this period, the three clones were able to asymptomatically invade the olfactory bulbs, brain, lungs and the middle ear mucosa and established populations in these tissues. The virulence potential of the three clones was poor even at high inoculum (105 CFU per mouse) concentrations in the mouse septicemia model and was undetectable in the pneumonia model. Capsular type 3 transformants of clones 6A and 19A prepared in the laboratory produced lethal infection at low cell concentration (103 CFU per mouse) but the same transformants became impaired in their potential to colonize, indicating the importance of the capsular polysaccharide in both disease and colonization. The three clones were compared to the genomes of 56 S. pneumoniae strains for which sequence information was available in the public databank. Clone 15A (ST63) only differed from the serotype 19F clone G54 in a very few genes including serotype so that this clone may be considered the product of a capsular switch. While no strain with comparable degree of similarity to clone 19A (ST276) was found among the sequenced isolates, by MLST this clone is a single locust variant (SLV) of Denmark14-ST230 international clone. Clone 6A (ST2191) was most similar to the penicillin resistant Hungarian serotype 19A clone.
doi:10.1371/journal.pone.0074867
PMCID: PMC3777985  PMID: 24069360
8.  Epidemiology and Population Structure of Serotypes 1, 5 and 7F Carried by Children in Portugal from 1996-2010 before Introduction of the 10-Valent and 13-Valent Pneumococcal Conjugate Vaccines 
PLoS ONE  2013;8(9):e75442.
Among the over 90 serotypes of Streptococcus pneumoniae described, serotypes 1, 5, and 7F account for a significant proportion of invasive disease worldwide and are now covered by the most recent 10- and 13-valent pneumococcal conjugate vaccines (PCVs). The epidemiology of these serotypes in carriage remains poorly studied because they are rarely detected. We aimed to gain insights into the epidemiology and population structure of serotypes 1, 5 and 7F carried by children in Portugal before PCV10 and PCV13 became widely used. Isolates obtained in cross-sectional studies carried out over a 15-year period (1996–2010) were retrospectively pooled and characterized. Of 5,123 pneumococci obtained, 70 were associated with serotypes 1 (n = 21), 5 (n = 7), and 7F (n = 42). The highest prevalence detected was 3.3% for serotype 1 in 2006, 1% for serotype 5 in 2009, and 3.3% for serotype 7F in 2006; Serotype 1 was associated with PMEN international clones Sweden1-28(ST306) and Sweden1-40(ST304); serotype 5 was associated with Colombia5-19(ST289); and serotype 7F was associated with Netherlands7F-39(ST191). All these isolates were fully susceptible. Most carriers of serotypes 1 (86%), 5 (86%), and 7F (91%) were older than two years but a significant association with older age was only observed for serotype 7F (p = 0.006). Evidence for cross-transmission was obtained. In conclusion, we were able to detect and characterize the rarely carried serotypes 1, 5, and 7F among healthy children in Portugal. These data will constitute an important baseline for upcoming surveillance studies aimed to establish the impact of novel PCVs targeting these serotypes in carriage.
doi:10.1371/journal.pone.0075442
PMCID: PMC3776787  PMID: 24058686
9.  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
11.  Disease Isolates of Streptococcus pseudopneumoniae and Non-Typeable S. pneumoniae Presumptively Identified as Atypical S. pneumoniae in Spain 
PLoS ONE  2013;8(2):e57047.
We aimed to obtain insights on the nature of a collection of isolates presumptively identified as atypical Streptococcus pneumoniae recovered from invasive and non-invasive infections in Spain. One-hundred and thirty-two isolates were characterized by: optochin susceptibility in ambient and CO2-enriched atmosphere; bile solubility; PCR-based assays targeting pneumococcal genes lytA, ply, pspA, cpsA, Spn9802, aliB-like ORF2, and a specific 16S rRNA region; multilocus sequence analysis; and antimicrobial susceptibility. By multilocus sequence analysis, 61 isolates were S. pseudopneumoniae, 34 were pneumococci, 13 were S. mitis, and 24 remained unclassified as non-pneumococci. Among S. pseudopneumoniae isolates, 51 (83.6%) were collected from respiratory tract samples; eight isolates were obtained from sterile sources. High frequency of non-susceptibility to penicillin (60.7%) and erythromycin (42.6%) was found. Only 50.8% of the S. pseudopneumoniae isolates displayed the typical optochin phenotype originally described for this species. None harbored the cpsA gene or the pneumococcal typical lytA restriction fragment length polymorphism. The Spn9802 and the specific 16S rRNA regions were detected among the majority of the S. pseudopneumoniae isolates (n = 59 and n = 49, respectively). The ply and pspA genes were rarely found. A high genetic diversity was found and 59 profiles were identified. Among the S. pneumoniae, 23 were capsulated and 11 were non-typeable. Three non-typeable isolates, associated to international non-capsulated lineages, were recovered from invasive disease sources. In conclusion, half of the atypical pneumococcal clinical isolates were, in fact, S. pseudopneumoniae and one-fourth were other streptococci. We identified S. pseudopneumoniae and non-typeable pneumococci as cause of disease in Spain including invasive disease.
doi:10.1371/journal.pone.0057047
PMCID: PMC3578818  PMID: 23437306
12.  Properties of a Novel PBP2A Protein Homolog from Staphylococcus aureus Strain LGA251 and Its Contribution to the β-Lactam-resistant Phenotype* 
The Journal of Biological Chemistry  2012;287(44):36854-36863.
Background: The recently isolated MRSA LGA251 has low resistance and carries a new mecA homolog.
Results: PBP2ALGA, the protein product of the new mecA, showed a “preference” for penicillins and instability at 37 °C. mecALGA251 introduced into susceptible S. aureus allowed expression of high-level resistance.
Significance: This study provides insights into the relationship between structure and function of PBP2A-like proteins.
Methicillin-resistant Staphylococcus aureus (MRSA) strains show strain-to-strain variation in resistance level, in genetic background, and also in the structure of the chromosomal cassette (SCCmec) that carries the resistance gene mecA. In contrast, strain-to-strain variation in the sequence of the mecA determinant was found to be much more limited among MRSA isolates examined so far. The first exception to this came with the recent identification of MRSA strain LGA251, which carries a new homolog of this gene together with regulatory elements mecI/mecR that also have novel, highly divergent structures. After cloning and purification in Escherichia coli, PBP2ALGA, the protein product of the new mecA homolog, showed aberrant mobility in SDS-PAGE, structural instability and loss of activity at 37 °C, and a higher relative affinity for oxacillin as compared with cefoxitin. The mecA homolog free of its regulatory elements was cloned into a plasmid and introduced into the background of the β-lactam-susceptible S. aureus strain COL-S. In this background, the mecA homolog expressed a high-level resistance to cefoxitin (MIC = 400 μg/ml) and a somewhat lower resistance to oxacillin (minimal inhibitory concentration = 200 μg/ml). Similar to PBP2A, the protein homolog PBP2ALGA was able to replace the essential function of the S. aureus PBP2 for growth. In contrast to PBP2A, PBP2ALGA did not depend on the transglycosylase activity of the native PBP2 for expression of high level resistance to oxacillin, suggesting that the PBP2A homolog may preferentially cooperate with a monofunctional transglycosylase as the alternative source of transglycosylase activity.
doi:10.1074/jbc.M112.395962
PMCID: PMC3481288  PMID: 22977239
Antibiotic Resistance; Antibiotics; Circular Dichroism (CD); Staphylococcus Aureus; Western Blotting; MRSA; Homologs of mecA and Penicillin-binding Protein (PBP)2A
13.  The Anti-Repressor MecR2 Promotes the Proteolysis of the mecA Repressor and Enables Optimal Expression of β-lactam Resistance in MRSA 
PLoS Pathogens  2012;8(7):e1002816.
Methicillin-resistant Staphylococcus aureus (MRSA) is an important human pathogen, which is cross-resistant to virtually all β-lactam antibiotics. MRSA strains are defined by the presence of mecA gene. The transcription of mecA can be regulated by a sensor-inducer (MecR1) and a repressor (MecI), involving a unique series of proteolytic steps. The induction of mecA by MecR1 has been described as very inefficient and, as such, it is believed that optimal expression of β-lactam resistance by MRSA requires a non-functional MecR1-MecI system. However, in a recent study, no correlation was found between the presence of functional MecR1-MecI and the level of β-lactam resistance in a representative collection of epidemic MRSA strains. Here, we demonstrate that the mecA regulatory locus consists, in fact, of an unusual three-component arrangement containing, in addition to mecR1-mecI, the up to now unrecognized mecR2 gene coding for an anti-repressor. The MecR2 function is essential for the full induction of mecA expression, compensating for the inefficient induction of mecA by MecR1 and enabling optimal expression of β-lactam resistance in MRSA strains with functional mecR1-mecI regulatory genes. Our data shows that MecR2 interacts directly with MecI, destabilizing its binding to the mecA promoter, which results in the repressor inactivation by proteolytic cleavage, presumably mediated by native cytoplasmatic proteases. These observations point to a revision of the current model for the transcriptional control of mecA and open new avenues for the design of alternative therapeutic strategies for the treatment of MRSA infections. Moreover, these findings also provide important insights into the complex evolutionary pathways of antibiotic resistance and molecular mechanisms of transcriptional regulation in bacteria.
Author Summary
Methicillin-resistance Staphylococcus aureus (MRSA) is an important human pathogen, causing a wide range of infections. MRSA strains are resistant to virtually all β-lactam antibiotics and often are also resistant to many other classes of antibiotics, leaving physicians with few therapeutic options. MRSA is defined by the presence of the mecA gene. The induction of mecA transcription in response to β-lactams involves a unique series of proteolytic steps and some critical details of this signal transduction mechanism are still illusive. For instance, it is not fully explained why the induction of mecA by its cognate regulatory genes mecR1-mecI appears to be very inefficient and it is not clear if the observed MecI repressor proteolysis is mediated directly by the activated MecR1 sensor-inducer. In this study, we demonstrate that the mecA regulatory locus is not a two-component system but instead it is a three-component system containing the previously unrecognized anti-repressor mecR2 gene. MecR2 disturbs the binding of the repressor MecI to the mecA promoter, which leads to its proteolytic inactivation independently from MecR1. Moreover, our data shows that in the presence of functional mecR1-mecI genes, mecR2 is essential for a robust induction of mecA transcription and, as consequence, for the optimal expression of β-lactam resistance.
doi:10.1371/journal.ppat.1002816
PMCID: PMC3406092  PMID: 22911052
14.  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
15.  Clonal Evolution Leading to Maintenance of Antibiotic Resistance Rates among Colonizing Pneumococci in the PCV7 Era in Portugal ▿  
Journal of Clinical Microbiology  2011;49(8):2810-2817.
The introduction of the seven-valent pneumococcal conjugate vaccine (PCV7) in Portugal led to extensive serotype replacement among carriers of pneumococci, with a marked decrease of PCV7 types. Although antimicrobial resistance was traditionally associated with PCV7 types, no significant changes in the rates of nonsusceptibility to penicillin, resistance to macrolides, or multidrug resistance were observed. This study aimed to investigate the mechanisms leading to maintenance of antimicrobial resistance, despite marked serotype replacement. We compared, through molecular typing, 252 antibiotic-resistant pneumococci recovered from young carriers in 2006 and 2007 (era of high PCV7 uptake) with collections of isolates from 2002 and 2003 (n=374; low-PCV7-uptake era) and 1996 to 2001 (n=805; pre-PCV7 era). We observed that the group of clones that has accounted for antimicrobial resistance since 1996 is essentially the same as the one identified in the PCV7 era. The relative proportions of such clones have, however, evolved substantially overtime. Notably, widespread use of PCV7 led to an expansion of two Pneumococcal Molecular Epidemiology Network (PMEN) clones expressing non-PCV7 capsular variants of the original strains: Sweden15AST63 (serotypes 15A and 19A) and Denmark14ST230 (serotypes 19A and 24F). These variants were already in circulation in the pre-PCV7 era, although they have now become increasingly abundant. Emergence of novel clones and de novo acquisition of resistance contributed little to the observed scenario. No evidence of capsular switch events occurring after PCV7 introduction was found. In the era of PCVs, antimicrobial resistance remains a problem among the carried pneumococci. Continuous surveillance is warranted to evaluate serotype and clonal shifts leading to maintenance of antimicrobial resistance.
doi:10.1128/JCM.00517-11
PMCID: PMC3147772  PMID: 21632898
16.  Identification of Genetic Determinants and Enzymes Involved with the Amidation of Glutamic Acid Residues in the Peptidoglycan of Staphylococcus aureus 
PLoS Pathogens  2012;8(1):e1002508.
The glutamic acid residues of the peptidoglycan of Staphylococcus aureus and many other bacteria become amidated by an as yet unknown mechanism. In this communication we describe the identification, in the genome of S. aureus strain COL, of two co-transcribed genes, murT and gatD, which are responsible for peptidoglycan amidation. MurT and GatD have sequence similarity to substrate-binding domains in Mur ligases (MurT) and to the catalytic domain in CobB/CobQ-like glutamine amidotransferases (GatD). The amidation of glutamate residues in the stem peptide of S. aureus peptidoglycan takes place in a later step than the cytoplasmic phase – presumably the lipid phase - of the biosynthesis of the S. aureus cell wall precursor. Inhibition of amidation caused reduced growth rate, reduced resistance to beta-lactam antibiotics and increased sensitivity to lysozyme which inhibited culture growth and caused degradation of the peptidoglycan.
Author Summary
Genetic determinants and enzymes that catalyze the multiple steps in the assembly of bacterial cell wall peptidoglycan have been known for some time. On the other hand, the mechanism by which glutamic acid residues of this structure undergo modification to glutamine has remained unknown. In this communication, we describe the identification of two genetic determinants that appear to be responsible for the completion of the chemical structure of the cell wall of the important human pathogen S. aureus. The availability of a conditional mutant which allows modulation of this system has allowed us to recognize the importance of glutamine residues for optimal growth rate and drug resistance and sensitivity of the staphylococcal peptidoglycan to the host defense factor lysozyme.
doi:10.1371/journal.ppat.1002508
PMCID: PMC3267633  PMID: 22303291
17.  Decrease in Pneumococcal Co-Colonization following Vaccination with the Seven-Valent Pneumococcal Conjugate Vaccine 
PLoS ONE  2012;7(1):e30235.
Understanding the epidemiology of pneumococcal co-colonization is important for monitoring vaccine effectiveness and the occurrence of horizontal gene transfer between pneumococcal strains. In this study we aimed to evaluate the impact of the seven-valent pneumococcal conjugate vaccine (PCV7) on pneumococcal co-colonization among Portuguese children. Nasopharyngeal samples from children up to 6 years old yielding a pneumococcal culture were clustered into three groups: pre-vaccine era (n = 173), unvaccinated children of the vaccine era (n = 169), and fully vaccinated children (4 doses; n = 150). Co-colonization, serotype identification, and relative serotype abundance were detected by analysis of DNA of the total bacterial growth of the primary culture plate using the plyNCR-RFLP method and a molecular serotyping microarray-based strategy. The plyNCR-RFLP method detected an overall co-colonization rate of 20.1%. Microarray analysis confirmed the plyNCR-RFLP results. Vaccination status was the only factor found to be significantly associated with co-colonization: co-colonization rates were significantly lower (p = 0.004; Fisher's exact test) among fully vaccinated children (8.0%) than among children from the pre-PCV7 era (17.3%) or unvaccinated children of the PCV7 era (18.3%). In the PCV7 era there were significantly less non-vaccine type (NVT) co-colonization events than would be expected based on the NVT distribution observed in the pre-PCV7 era (p = 0.024). In conclusion, vaccination with PCV7 resulted in a lower co-colonization rate due to an asymmetric distribution between NVTs found in single and co-colonized samples. We propose that some NVTs prevalent in the PCV7 era are more competitive than others, hampering their co-existence in the same niche. This result may have important implications since a decrease in co-colonization events is expected to translate in decreased opportunities for horizontal gene transfer, hindering pneumococcal evolution events such as acquisition of antibiotic resistance determinants or capsular switch. This might represent a novel potential benefit of conjugate vaccines.
doi:10.1371/journal.pone.0030235
PMCID: PMC3257259  PMID: 22253924
18.  Analysis of Invasiveness of Pneumococcal Serotypes and Clones Circulating in Portugal before Widespread Use of Conjugate Vaccines Reveals Heterogeneous Behavior of Clones Expressing the Same Serotype▿† 
Journal of Clinical Microbiology  2011;49(4):1369-1375.
To estimate the invasive disease potential of serotypes and clones circulating in Portugal before extensive use of the seven-valent pneumococcal conjugate vaccine, we analyzed 475 invasive isolates recovered from children and adults and 769 carriage isolates recovered from children between 2001 and 2003. Isolates were serotyped and genotyped by pulsed-field gel electrophoresis, and a selection of isolates were also characterized by multilocus sequence typing. We found that the diversities of serotypes and genotypes of pneumococci responsible for invasive infections and carriage were identical and that most carried clones could also be detected as causes of invasive disease. Their ability to do so, however, varied substantially. Serotypes 1, 3, 4, 5, 7F, 8, 9N, 9L, 12B, 14, 18C, and 20 were found to have an enhanced propensity to cause invasive disease, while serotypes 6A, 6B, 11A, 15B/C, 16F, 19F, 23F, 34, 35F, and 37 were associated with carriage. In addition, significant differences in invasive disease potential between clones sharing the same serotype were found among several serotypes, namely, 3, 6A, 6B, 11A, 14, 19A, 19F, 22F, 23F, 34, and NT. This heterogeneous behavior of the clones was found irrespective of the serotype's overall invasive disease potential. Our results highlight the importance of the genetic background when analyzing the invasive disease potential of certain serotypes and provide an important baseline for its monitoring following conjugate vaccine use. Continuous surveillance should be maintained, and current research should focus on uncovering the genetic determinants that contribute to the heterogeneity of invasive disease potential of clones sharing the same serotype.
doi:10.1128/JCM.01763-10
PMCID: PMC3122870  PMID: 21270219
19.  Methicillin-Resistance in Staphylococcus aureus Is Not Affected by the Overexpression in Trans of the mecA Gene Repressor: A Surprising Observation 
PLoS ONE  2011;6(8):e23287.
Methicillin-resistant Staphylococcus aureus (MRSA) is intrinsically cross-resistant to virtually all β-lactam antibiotics. The central determinant for the MRSA phenotype is the mecA gene, whose transcriptional control may be mediated by a repressor (mecI) and a sensor/inducer (mecR1). The mecI-mecR1-mediated induction of mecA takes several hours rendering the strains phenotypically susceptible in spite of the presence of the resistance gene. Therefore, it has been proposed that the full resistance to β-lactams observed in many contemporary clinical MRSA strains requires a non-functional mecI-mecR1 regulatory system. The mecA gene is embedded in a large chromosomal cassette (the SCCmec element) for which several structural types have been described. Some epidemic MRSA clones, typically expressing full β-lactam resistance, carry SCCmec elements that contain an intact mecI-mecR1 locus (e.g. SCCmec types II and III). We have addressed this apparent contradiction by first sequencing the mecI coding region and mecA promoter sequences in a collection of prototype MRSA strains characterized by different SCCmec types. A conserved non-sense mutation within mecI was detected in all SCCmec type III strains tested, presumably responsible for a non-functional truncated MecI protein and, therefore, explaining the full resistance phenotype. In SCCmec type II strains no conserved mutations were found. We next transformed a collection of prototype MRSA epidemic strains with a recombinant plasmid overexpressing a wild-type copy of mecI. Surprisingly, for the great majority of the strains no significant alterations in the phenotypic expression of β-lactam resistance could be detected. These findings were confirmed and further explored, challenging the currently accepted mechanism of mecA transcriptional control. Our observations suggest the existence of yet unidentified additional determinants involved in the transcriptional control of mecA gene and point to a revision of the mecA regulatory mechanism in contemporary MRSA strains.
doi:10.1371/journal.pone.0023287
PMCID: PMC3149077  PMID: 21829724
20.  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
21.  Evidence for a purifying selection acting on the β-lactamase locus in epidemic clones of methicillin-resistant Staphylococcus aureus 
BMC Microbiology  2011;11:76.
Background
The β-lactamase (bla) locus, which confers resistance to penicillins only, may control the transcription of mecA, the central element of methicillin resistance, which is embedded in a polymorphic heterelogous chromosomal cassette (the SCCmec element). In order to assess the eventual correlation between bla allotypes and genetic lineages, SCCmec types and/or β-lactam resistance phenotypes, the allelic variation on the bla locus was evaluated in a representative collection of 54 international epidemic methicillin-resistant Staphylococcus aureus (MRSA) clinical strains and, for comparative purposes, also in 24 diverse methicillin-susceptible S. aureus (MSSA) strains.
Results
Internal fragments of blaZ (the β-lactamase structural gene) were sequenced for all strains. A subset of strains, representative of blaZ allotypes, was further characterized by sequencing of internal fragments of the blaZ transcriptional regulators, blaI and blaR1. Thirteen allotypes for blaZ, nine for blaI and 12 for blaR1 were found. In a total of 121 unique single-nucleotide polymorphisms (SNP) detected, no frameshift mutations were identified and only one nonsense mutation within blaZ was found in a MRSA strain. On average, blaZ alleles were more polymorphic among MSSA than in MRSA (14.7 vs 11.4 SNP/allele). Overall, blaR1 was the most polymorphic gene with an average of 24.8 SNP/allele. No correlation could be established between bla allotypes and genetic lineages, SCCmec types and/or β-lactam resistance phenotypes. In order to estimate the selection pressure acting on the bla locus, the average dN/dS values were computed. In the three genes and in both collections dN/dS ratios were significantly below 1.
Conclusions
The data strongly suggests the existence of a purifying selection to maintain the bla locus fully functional even on MRSA strains. Although, this is in agreement with the notion that in most clinical MRSA strains mecA gene is under the control of the bla regulatory genes, these findings also suggest that the apparently redundant function of blaZ gene for the MRSA resistant phenotype is still important for these strains. In addition, the data shows that the sensor-inducer blaR1 is the primary target for the accumulation of mutations in the bla locus, presumably to modulate the response to the presence of β-lactam antibiotic.
doi:10.1186/1471-2180-11-76
PMCID: PMC3102608  PMID: 21496235
β-lactamase; β-lactam resistance; allelic variation; MSSA; MRSA; mecA stabilization
22.  High Prevalence of EMRSA-15 in Portuguese Public Buses: A Worrisome Finding 
PLoS ONE  2011;6(3):e17630.
Background
The nosocomial prevalence of methicillin resistant Staphylococcus aureus (MRSA) in Portugal remains one of the highest in Europe and is currently around 50%. Transmission of S. aureus, including MRSA, occurs principally by direct human-to-human skin contact. However, S. aureus can survive for long periods on inanimate objects, which may represent an important reservoir for dissemination as well.
Methodology/Principal Findings
Between May 2009 and February 2010, handrails of 85 public urban buses circulating in Oporto, Portugal, were screened for the occurrence of MRSA. Twenty-two (26%) buses showed MRSA contamination. The molecular characterization of a total of 55 MRSA, by pulsed-field gel electrophoresis (PFGE), staphylococcal cassette chromosome (SCC) mec typing, spa typing, and multilocus sequence typing (MLST), clustered the isolates into three clonal types. However, the overwhelming majority (n = 50; 91%) of the isolates belonged to a single clone (PFGE A, spa types t747, t032, t025 or t020, ST22, SCCmec type IVh) that exhibits the characteristics of the pandemic EMRSA-15, currently the major lineage circulating in Portuguese hospitals, namely in the Oporto region. Two additional clones were found but in much lower numbers: (i) PFGE B, ST5, spa type t002, SCCmec IVa (n = 3), and (ii) PFGE C, spa type t008, ST8, SCCmec IVa (n = 2). None of the 55 isolates was PVL positive.
Conclusions/Significance
Public buses in Oporto seem to be an important reservoir of MRSA of nosocomial origin, providing evidence that the major hospital-associated MRSA clone in Portugal is escaping from the primary ecological niche of hospitals to the community environment. Infection control measures are urgently warranted to limit the spread of EMRSA-15 to the general population and future studies are required to assess the eventual increase of MRSA in the Portuguese community, which so far remains low.
doi:10.1371/journal.pone.0017630
PMCID: PMC3047573  PMID: 21407807
23.  Evolution of MRSA During Hospital Transmission and Intercontinental Spread 
Science (New York, N.Y.)  2010;327(5964):469-474.
Current methods for differentiating isolates of predominant lineages of pathogenic bacteria often do not provide sufficient resolution to define precise relationships. Here, we describe a high-throughput genomics approach that provides a high-resolution view of the epidemiology and microevolution of a dominant strain of methicillin-resistant Staphylococcus aureus (MRSA). This approach reveals the global geographic structure within the lineage, its intercontinental transmission through four decades, and the potential to trace person-to-person transmission within a hospital environment. The ability to interrogate and resolve bacterial populations is applicable to a range of infectious diseases, as well as microbial ecology.
doi:10.1126/science.1182395
PMCID: PMC2821690  PMID: 20093474
24.  Denmark14-230 Clone as an Increasing Cause of Pneumococcal Infection in Portugal within a Background of Diverse Serotype 19A Lineages▿  
Journal of Clinical Microbiology  2009;48(1):101-108.
Pneumococci of serotype 19A are increasingly found to be the cause of infection in various geographic regions. We have characterized the serotype 19A isolates (n = 288) found among pneumococci responsible for infections (n = 1,925) and pneumococci recovered from asymptomatic carriers (n = 1,973) in Portugal between 2001 and 2006. We show that despite the existence of serotype 19A clones that have a greater potential to cause invasive disease or an enhanced colonization capacity, the lineage that is increasing as a cause of infection in Portugal is a multiresistant clone that is competent at both. The expanding Denmark14-230 clone found in Portugal is disseminated in other Mediterranean countries, where it is also increasingly responsible for invasive infections in both children and adults. The lineages driving the rise of serotype 19A infections in Asia and the United States (sequence type 320 [ST320] and ST199) are either absent or account for only a small proportion of isolates in Portugal. These data highlight the importance of locally circulating clones with the ability to compete in the nasopharyngeal niche in the emergence of the serotype 19A lineages which are an increasing cause of infection in various geographic regions.
doi:10.1128/JCM.00665-09
PMCID: PMC2812288  PMID: 19864476
25.  Molecular Epidemiology and Risk Factors for Nasal Carriage of Staphylococcus aureus and Methicillin-Resistant S. aureus in Infants Attending Day Care Centers in Brazil▿  
Journal of Clinical Microbiology  2009;47(12):3991-3997.
Investigations regarding Staphylococcus aureus carriage among Brazilian children are scarce. We evaluated the determinants of S. aureus and methicillin-resistant S. aureus (MRSA) nasal carriage in infants attending day care centers (DCCs) and the molecular features of the MRSA strains. A total of 1,192 children aged 2 months to 5 years attending 62 DCCs were screened for S. aureus and MRSA nasal carriage. MRSA isolates were characterized by pulsed-field gel electrophoresis, multilocus sequence typing, spa typing, staphylococcal cassette chromosome (SCC) mec typing and the presence of the Panton-Valentine leukocidin gene. Logistic regression was performed to determine risk factors associated with S. aureus and MRSA colonization. S. aureus and MRSA carriage were detected in 371 (31.1%) and 14 (1.2%) children, respectively. Variables found to be independently associated with an increased risk for S. aureus carriage included being older than 24 months (odds ratio [OR], 1.8; 95% confidence interval [CI], 1.3 to 2.6) and previous DCC attendance (OR, 1.5; 95% CI, 1.0 to 2.2). Having a mother with a high level of education was a protective factor for nasal colonization (OR, 0.4; 95% CI, 0.2 to 0.8). Moreover, we observed that more children carrying MRSA had younger siblings than children not colonized by MRSA. Among the 14 MRSA strains, three SCCmec types (IIIA, IV, and V) were detected, together with a multidrug-resistant dominant MRSA lineage sharing 82.7% genetic similarity with the Brazilian clone (ST239-MRSA-IIIA; ST indicates the sequence type determined by multilocus sequence typing). Although SCCmec type V was recovered from one healthy child who had been exposed to known risk factors for hospital-associated MRSA, its genetic background was compatible with community-related MRSA. Our data suggest that DCC attendees could be contributing to MRSA cross-transmission between health care and community settings.
doi:10.1128/JCM.01322-09
PMCID: PMC2786686  PMID: 19828745

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