Ana Friães

Novel Role of the Antimicrobial Peptide LL-37 in the Protection of Neutrophil Extracellular Traps against Degradation by Bacterial Nucleases

Neutrophil extracellular traps (NETs) have been described as a fundamental innate immune defence mechanism. They consist of a nuclear DNA backbone associated with different antimicrobial peptides (AMPs) which are able to engulf and kill pathogens. The AMP LL-37, a member of the cathelicidin family, is highly present in NETs. However, the function of LL-37 within NETs is still unknown because it loses its antimicrobial activity when bound to DNA in the NETs. Using immunofluorescence microscopy, we demonstrate that NETs treated with LL-37 are distinctly more resistant to S. aureus nuclease degradation than nontreated NETs. Biochemical assays utilising a random LL-37-fragment library indicated that the blocking effect of LL-37 on nuclease activity is based on the cationic character of the AMP, which facilitates the binding to neutrophil DNA, thus protecting it from degradation by the nuclease. In good correlation to these data, the cationic AMPs human beta defensin-3 and human neutrophil peptide-1 showed similar protection of neutrophil-derived DNA against nuclease degradation. In conclusion, this study demonstrates a novel role of AMPs in host immune defence: beside its direct antimicrobial activity against various pathogens, cationic AMPs can stabilise neutrophil-derived DNA or NETs against bacterial nuclease degradation.

Group A streptococci clones associated with invasive infections and pharyngitis in Portugal present differences in emm types, superantigen gene content and antimicrobial resistance

BackgroundA few lineages of Group A streptococci (GAS) have been associated with a reemergence of severe invasive streptococcal disease in developed countries. However, the majority of the comparisons between invasive and non-invasive GAS isolates have been performed for collections of reduced genetic diversity or relied on limited typing information to distinguish clones. We characterized by several typing methods and compared a collection of 160 isolates recovered from normally sterile sites with 320 isolates associated with pharyngitis and recovered in the same time period in Portugal.ResultsAlthough most of the isolates belonged to clones that were equally prevalent in invasive infections and pharyngitis, we identified markers of invasiveness, namely the emm types 1 and 64, and the presence of the speA and speJ genes. In contrast, emm4, emm75, and the ssa and speL/M genes were significantly associated with pharyngitis. There was a strong agreement between the emm type, the superantigen (SAg) genes and the clusters defined by pulsed-field gel electrophoresis (PFGE) profiling. Therefore, combinations of particular emm types and SAg genes frequently co-occurred in the same PFGE cluster, but there was no synergistic or antagonistic interaction between them in determining invasiveness. Only macrolide-susceptible PFGE clones were significantly associated with invasive infections or pharyngitis, while the clones of resistant isolates sharing all other molecular properties analyzed were equally prevalent in the two groups of isolates.ConclusionsThis study confirmed the importance of the widely disseminated emm1-T1-ST28 clone in invasive infections but also identified other clones linked to either invasive infections (emm64-ST164) or pharyngitis (emm4-T4-ST39), which may be more limited in their temporal and geographical spread. Clonal properties like some emm types or SAg genes were associated with disease presentation, highlighting the importance of bacterial genetic factors to the outcome of GAS infections, although other, yet unidentified factors may also play an important role.

Changes in Streptococcus pyogenes causing invasive disease in Portugal: Evidence for superantigen gene loss and acquisition

The emergence of highly virulent and successful Streptococcus pyogenes (group A streptococci - GAS) clones has been attributed to the exchange of virulence factors by lateral gene transfer mechanisms, which strongly contribute to genomic diversity. We characterized a collection of 191 GAS isolates recovered from normally sterile sites in Portugal during 2006-2009 and compared them to invasive isolates obtained during 2000-2005. Antimicrobial resistance rates did not change significantly between the two periods and were generally low. In 2006-2009, emm1, emm89, emm3, and emm6 represented 60% of the isolates. The chromosomally encoded superantigen (SAg) genes speG and smeZ were present in the majority (>90%) of the isolates, while speJ was found in only 45%. The phage encoded SAgs varied greatly in prevalence (2-53%). The distribution of emm types, pulsed-field gel electrophoresis profiling (PFGE) clusters, and SAg profiles changed significantly between the periods, although there were no statistically supported changes in the prevalence of individual types. While the macrolide susceptible clone emm1-T1-ST28 remained dominant (28%), there was a significant decrease in clonal diversity as indicated by both PFGE profiling and emm typing. This was accompanied by intra-clonal divergence of SAg profiles, which was statistically confirmed for isolates representing emm1, emm28, and emm44. This diversification was associated with the loss and acquisition of SAg genes, carried by phages and of chromosomal origin. These data suggest an ongoing genomic diversification of GAS invasive isolates in Portugal that may contribute to the persistence of clones with improved fitness or virulence.

Differences between Macrolide-Resistant and -Susceptible Streptococcus pyogenes: Importance of Clonal Properties in Addition to Antibiotic Consumption

ABSTRACT A steady decline in macrolide resistance among Streptococcus pyogenes (group A streptococci [GAS]) in Portugal was reported during 1999 to 2006. This was accompanied by alterations in the prevalence of macrolide resistance phenotypes and in the clonal composition of the population. In order to test whether changes in the macrolide-resistant population reflected the same changing patterns of the overall population, we characterized both macrolide-susceptible and -resistant GAS associated with a diagnosis of tonsillo-pharyngitis recovered in the period from 2000 to 2005 in Portugal. Pulsed-field gel electrophoresis (PFGE) profiling was the best predictor of emm type and the only typing method that could discriminate clones associated with macrolide resistance and susceptibility within each emm type. Six PFGE clusters were significantly associated with macrolide susceptibility: T3-emm3-ST406, T4-emm4-ST39, T1-emm1-ST28, T6-emm6-ST382, B3264-emm89-ST101/ST408, and T2-emm2-ST55. Four PFGE clusters were associated with macrolide resistance: T4-emm4-ST39, T28-emm28-ST52, T12-emm22-ST46, and T1-emm1-ST28. We found no evidence for frequent ongoing horizontal transfer of macrolide resistance determinants. The diversity of the macrolide-resistant population was lower than that of susceptible isolates. The differences found between the two populations suggest that the macrolide-resistant population of GAS has its own dynamics, independent of the behavior of the susceptible population.

Epidemiological survey of the first case of vancomycin-resistant Staphylococcus aureus infection in Europe

We report on the follow-up and epidemiological study triggered by the isolation of the first vancomycin-resistant Staphylococcus aureus (VRSA) detected in Europe. The patient and 53 close contacts were screened for S. aureus colonization and all isolates recovered were characterized by multiple molecular typing methods. The VRSA remained confined to the infected foot of the patient and was not detected in any of the close contacts. Nasal colonization with S. aureus was detected in 20 subjects, of whom 15 carried methicilin-susceptible isolates with the remaining five harbouring methicilin-resistant S. aureus (MRSA). The majority of the isolates belonged to clones that have been previously shown to be prevalent in Portugal, both in the hospital setting and in the community. Only one isolate, an MRSA, was closely related to the VRSA. Like most of the characterized VRSA isolates from other countries, the VRSA isolated in Portugal belonged to clonal complex (CC) 5. Despite the absence of VRSA dissemination, the recent increase in the incidence of lineages belonging to CC5 in some European countries, including Portugal, may result in more frequent opportunities for the emergence of VRSA.

Macrolide-resistant Streptococcus pyogenes: prevalence and treatment strategies

Although penicillin remains the first-choice treatment for Streptococcus pyogenes infection, macrolides are important alternatives for allergic patients and lincosamides are recommended together with β-lactams in invasive infections. S. pyogenes may exhibit macrolide resistance because of active efflux (mef genes) or target modification (erm genes), the latter conferring cross resistance to lincosamides and streptogramin B. Worldwide, resistance is restricted to a limited number of genetic lineages, despite resistance genes being encoded on mobile genetic elements. For reasons that are not completely clear, resistance and the associated phenotypes are highly variable across countries. Although resistance remains high in several countries, particularly in Asia, an overall decreasing trend of resistance has been noted in recent years, mostly in Europe. This decrease is not always accompanied by declines in macrolide consumption, suggesting significant roles of other factors in determining the dynamics of macrolide-resistant clones. Continued surveillance is needed to obtain further insights into the forces governing macrolide resistance in S. pyogenes.

Consequences of the variability of the CovRS and RopB regulators among Streptococcus pyogenes causing human infections.

To evaluate the importance of covRS and ropB mutations in invasive disease caused by Group A Streptococci (GAS), we determined the sequence of the covRS and ropB genes of 191 isolates from invasive infections and pharyngitis, comprising a diverse set of emm types and multilocus sequence types. The production of SpeB and the activity of NAD glycohydrolase (NADase) and streptolysin S (SLS) were evaluated. The results support the acquisition of null covS alleles (predicted to eliminate protein function), resulting in downregulation of SpeB and upregulation of NADase and SLS, as a mechanism possibly contributing to higher invasiveness. Among the isolates tested, this mechanism was found to be uncommon (10% of invasive isolates) and was not more prevalent among clones with enhanced invasiveness (including M1T1) but occurred in diverse genetic backgrounds. In lineages such as emm64, these changes did not result in upregulation of NADase and SLS, highlighting the diversity of regulatory pathways in GAS. Despite abrogating SpeB production, null alleles in ropB were not associated with invasive infection. The covRS and ropB genes are under stabilising selection and no expansion of isolates carrying null alleles has been observed, suggesting that the presence of these regulators is important for overall fitness.

Emergence of the Same Successful Clade among Distinct Populations of emm89 Streptococcus pyogenes in Multiple Geographic Regions

The emergence of clades within emm89 Streptococcus pyogenes isolates that rapidly became the dominant lineages expressing this emm type was recently reported in the United Kingdom (1) and in a study that included isolates from the United States, Finland, and Iceland (United States/FI/IC) (2). In the United Kingdom, the emerging clade was associated with the absence of the hasABC locus, responsible for the synthesis of the hyaluronic acid capsule (1). The study from the United States/FI/IC (2) highlighted the strict association of the emerging clade with an nga promoter variant, also found in contemporary emm1 isolates, which results in increased expression of the nga locus. The study from the United Kingdom also examined this region and found thatthenga-ifs-slolocusandsurroundingsequencesoftheemerging clade shared 99% DNA identity with that of contemporary emm1 and emm12 strains, but the authors do not offer any information on thengapromoter (1). The acquisition of this region by emm1 isolates is currently considered the major molecular event triggering the success and enhanced virulence of this clone (3). Given that the two studies characterized emm89 strains recoveredinoverlappingtimeperiods,itwaspossiblethatthetwowere documentingthedisseminationofthesamecladeindifferentgeographic areas, although the information presented did not allow this conclusion since the papers analyze different aspects of the strains. We set out to test this hypothesis by reanalyzing the publicly available data, and we also characterized emm89 isolates recoveredinPortugaltoinvestigateifthiscladecouldbealsoemerging in southern Europe. While analyzing this data, an additional paperwaspublishedfocusingontheinterplaybetweenexpression of the nga-ifs-slo locus and that of capsule in the virulence of the emm89 strains of the United States/FI/IC study (4). We analyzed the sequencing reads deposited in public databases from the strains included in the two papers in order to determine the presence of the hasABC locus, the sequence types (STs), the variant of the nga promoter, and possible variations in the nga coding sequence. Briefly, the raw sequencing reads were mapped using Bowtie2 to exemplar sequences of each of the loci together with at least 600 bp of upstream and downstream sequence. A total of 907 strains met the quality standards that we required in our analysis in both the has and nga loci. Multilocus sequence typing (MLST) alleles could be confidently determined for 886 of these strains. A total of 79 isolates presented novel STs due to the presence of new alleles or allelic profiles, which were submitted to the S. pyogenes MLST database (http://pubmlst.org/ spyogenes/) and assigned ST791 to ST804. In addition, we analyzed125emm89isolatesrecoveredinPortugalbetween2000and

Automatic determination of NET (neutrophil extracellular traps) coverage in fluorescent microscopy images

MOTIVATION Neutrophil extracellular traps (NETs) are believed to be essential in controlling several bacterial pathogens. Quantification of NETs in vitro is an important tool in studies aiming to clarify the biological and chemical factors contributing to NET production, stabilization and degradation. This estimation can be performed on the basis of fluorescent microscopy images using appropriate labelings. In this context, it is desirable to automate the analysis to eliminate both the tedious process of manual annotation and possible operator-specific biases. RESULTS We propose a framework for the automated determination of NET content, based on visually annotated images which are used to train a supervised machine-learning method. We derive several methods in this framework. The best results are obtained by combining these into a single prediction. The overall Q(2) of the combined method is 93%. By having two experts label part of the image set, we were able to compare the performance of the algorithms to the human interoperator variability. We find that the two operators exhibited a very high correlation on their overall assessment of the NET coverage area in the images (R(2) is 97%), although there were consistent differences in labeling at pixel level (Q(2), which unlike R(2) does not correct for additive and multiplicative biases, was only 89%). AVAILABILITY AND IMPLEMENTATION Open source software (under the MIT license) is available at https://github.com/luispedro/Coelho2015_NetsDetermination for both reproducibility and application to new data.

Pediatric Complicated Pneumonia Caused by Streptococcus pneumoniae Serotype 3 in 13-Valent Pneumococcal Conjugate Vaccinees, Portugal, 2010–2015

Despite use of 7-valent pneumococcal conjugate vaccine, incidence of pleural effusion and empyema (pediatric complicated pneumococcal pneumonia [PCPP]) is reportedly increasing globally. We cultured and performed PCR on 152 pleural fluid samples recovered from pediatric patients in Portugal during 2010–2015 to identify and serotype Streptococcus pneumoniae. We identified only 17 cases by culture, but molecular methods identified S. pneumoniae in 68% (92/135) of culture-negative samples. The most frequent serotypes were 3, 1, and 19A, together accounting for 62% (68/109) of cases. Nineteen cases attributable to 13-valent pneumococcal conjugate vaccine (PCV13) serotypes (mostly serotype 3) were detected among 22 children age-appropriately vaccinated with PCV13. The dominance of the additional serotypes included in PCV13 among PCPP cases in Portugal continues, even with PCV13 available on the private market (without reimbursement) since 2010 and with average annual coverage of 61% among age-eligible children. Our data suggest reduced effectiveness of PCV13 against serotype 3 PCPP.