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IntroductionNon-invasive pneumococcal pneumonia (NIPP) is possibly the most frequent infection by Streptococcus pneumoniae in adults. However, the herd effect of vaccinating children in adult NIPP (aNIPP) remains poorly characterized.MethodsWe determined the serotype distribution and antimicrobial susceptibility of isolates causing aNIPP (>18 years) in 2016–2018 in Portugal; 3 years with near universal vaccination of children with the 13-valent conjugate vaccine (PCV13), following over a decade of significant PCV use in children in the private market.Results and discussionAmong the 1,149 aNIPP isolates, the most frequent serotypes detected were: 3 (n = 168, 14.6%), 11A (n = 102, 8.9%), 19F (n = 70, 6.1%), 23A and 23B (n = 62, 5.4% each), 9N (n = 60, 5.2%), 8 and 29/35B (n = 43, 3.7% each); together accounting for 53% of all isolates. The serotype distribution causing aNIPP was stable in 2016–2018, with the serotypes included in PCV7 still being important causes of disease and serotype 3, a PCV13 serotype, remaining the leading cause of aNIPP. There was an increase in penicillin non-susceptibility from 17% in 2016 to 24% in 2018 (p = 0.018). Some PCV13 serotypes, such as 14, 19A and 19F were associated to resistance, which may have contributed to their persistence. The fact that close to 20% of aNIPP is caused by four non-vaccine serotypes (23A, 23B, 9N, and 29/35B) and that there were significant differences in serotype distribution relative to invasive disease, stress the importance of maintaining the surveillance of these infections. The lack of a continued herd effect from vaccinating children and the significant fraction of aNIPP potentially preventable by PCV13 (30%), PCV15 (34%), PCV20 (53%) and the 23-valent polysaccharide vaccine (61%) underscore the importance of considering the broader use of pneumococcal vaccines in adults.

The reasons why malaria manifests with a variety of well-recognized clinical phenotypes remain poorly understood. Here, using distinct rodent models, the authors reveal that the microbiota colonizing the lung promotes respiratory distress syndrome and mortality during malaria infections.

ABSTRACT The prevalence and lineages of Streptococcus agalactiae (group B streptococci [GBS]) colonizing pregnant women are well studied, but less is known about colonization of nonpregnant adults. We characterized GBS colonization in adults as a potential reservoir for infections and tested for the presence of clones with a potentially higher invasive disease potential. We evaluated GBS gastrointestinal, genitourinary, and oral colonization among 336 nonpregnant adults in the community. We characterized the isolates by serotyping, multilocus sequence typing, profiling of surface protein genes and pili, and antimicrobial susceptibility and compared them with contemporary invasive isolates. The colonization rate (n = 107, 32%) among nonpregnant adults was like that of pregnant women. Colonization increased with age (~25% in the 18 to 29 and 30 to 44 years old groups and >42% in the ≥60 years old group), potentially explaining the higher incidence of disease with older age. Participants who were colonized at multiple sites (73%) were frequently carrying indistinguishable strains (93%), consistent with the existence of a single reservoir of colonization and transfer of GBS between sites within the same individual. The most frequent lineages found were serotype Ib/CC1 (n = 27), serotype V/CC1 (n = 19), serotype Ia/CC23 (n = 13), serotype III/ST17 (n = 13), and serotype Ib/CC10 (n = 11). Comparison with contemporary isolates causing invasive infections in Portugal did not reveal any lineage associated with either asymptomatic carriage or invasive disease. Asymptomatic colonization of nonpregnant adults is significant and could act as a reservoir for invasive disease, but in contrast to infant disease, we found no GBS lineages with an enhanced potential for causing invasive disease in adults. IMPORTANCE The increasing incidence of Streptococcus agalactiae (group B streptococci [GBS]) infections in adults and the inability of antimicrobial prophylaxis peripartum to control late-onset infections in infants motivate the study of the asymptomatic carrier state in nonpregnant adults. We found an overall carriage rate like that of pregnant women, increasing with age, potentially contributing to the higher incidence of GBS infections with age. Colonization of diabetic participants was not higher despite the higher number of infections in this group. Comparison between contemporary genetic lineages causing infections and found in asymptomatic carriers did not identify particularly virulent lineages. This means that any prophylactic approaches targeting colonization by particular lineages are expected to have a limited impact on GBS disease in adults.

The introduction of pneumococcal conjugate vaccines PCV7 and PCV13 led to decreases in incidence of pediatric invasive pneumococcal disease (pIPD) and changes in serotype distribution. We evaluated the consequences of higher vaccine uptake after the introduction of PCV13 in the National Immunization Plan (NIP) in 2015. Besides culture and conventional serotyping, the use of molecular methods to detect and serotype pneumococci in both pleural and cerebrospinal fluid samples contributed to 30% of all pIPD (n = 232) in 2015–2018. The most frequently detected serotypes were: 3 (n = 59, 26%), 10A (n = 17, 8%), 8 (n = 16, 7%) and 19A (n = 10, 4%). PCV13 serotypes still accounted for 46% of pIPD cases. Serotypes not included in any currently available conjugate vaccine (NVT) are becoming important causes of pIPD, with the increases in serotypes 8 and 33F being of particular concern given the importance of serotype 8 in adult IPD and the antimicrobial resistance of serotype 33F isolates. This study highlights the importance of using molecular methods in pIPD surveillance since these allowed a better case ascertainment and the identification of serotype 3 as the leading cause of pIPD. Even in a situation of vaccine uptake >95% for 3 years, PCV13 serotypes remain important causes of pIPD.

Increasing the uptake of the 13-valent pneumococcal conjugate vaccine (PCV13) in children is expected to alter the serotypes causing invasive pneumococcal disease (IPD) in adults due to herd protection. We characterized 2172 cases of adult IPD in 2015–2018 in Portugal after the introduction of PCV13 in the national immunization plan of 2015. Among the 58 detected serotypes, serotypes 8 (n = 413; 19%), 3 (n = 334; 15%), 22F (n = 148; 7%), 14 (n = 138; 6%), and 19A (n = 116; 5%) were the most frequent. Among PCV13 serotypes, 7F and 19A IPD decreased, but serotype 3 IPD remained stable. The non-PCV13 serotypes were a heterogeneous group, with serotypes 23A and 23B enriched among CSF cases; serotype 8 associated with younger patients; and serotypes 22F, 6C, and 31 associated with older patients. The continued increase of serotype 8 IPD was one of the drivers for the increased coverage of the 23-valent pneumococcal polysaccharide vaccine (PPV23; 80% in 2015–2018). Antimicrobial resistance was associated with older age and serotypes 6C, 11A, 14, 15A, 19A, and 19F. Three years after the introduction of PCV13 in the NIP with an uptake of >95%, the proportion of PCV13 serotypes causing IPD in adults stabilized in Portugal. The direct vaccination of adults may be important in preventing IPD in this age group.

Streptococcus canis is an animal pathogen which occasionally causes infections in humans. The S. canis M-like protein (SCM) encoded by the scm gene, is its best characterized virulence factor but previous studies suggested it could be absent in a substantial fraction of isolates. We studied the distribution and variability of the scm gene in 188 S. canis isolates recovered from companion animals (n = 152), wild animal species (n = 20), and humans (n = 14). Multilocus sequence typing, including the first characterization of wildlife isolates, showed that the same lineages are present in all animal hosts, raising the possibility of extensive circulation between species. Whole-genome analysis revealed that emm-like genes found previously in S. canis correspond to divergent scm genes, indicating that what was previously believed to correspond to two genes is in fact the same scm locus. We designed primers allowing for the first time the successful amplification of the scm gene in all isolates. Analysis of the scm sequences identified 12 distinct types, which could be divided into two clusters: group I (76%, n = 142) and group II (24%, n = 46) sharing little sequence similarity. The predicted group I SCM showed extensive similarity with each other outside of the N-terminal hypervariable region and a conserved IgG binding domain. This domain was absent from group II SCM variants found in isolates previously thought to lack the scm gene, which also showed greater amino acid variability. Further studies are necessary to elucidate the possible host interacting partners of the group II SCM variants and their role in virulence.

Non-invasive pneumococcal pneumonia (NIPP) is a frequent cause of morbidity and mortality worldwide. The 13-valent pneumococcal conjugate vaccine (PCV13) was included in the national immunization program of children living in Portugal in 2015. Until then, PCV7 (since late 2001) and PCV13 (since early 2010) were given through the private market. We determined the serotype distribution and antimicrobial susceptibility of isolates causing adult NIPP in 2012-2015 and compared the results with previously published data (2007-2011). There were 50 serotypes among the 1435 isolates. The most common were serotypes: 3 (14%), 11A (8%), 19F (6%), 23A (5%), 6C (5%), 19A (4%), 23B (4%), 9N (4%) and non-typable isolates (4%). When considering data since the availability of PCV13 for children in the private market, the proportion of PCV13 serotypes declined from 44.0% in 2010 to 29.7% in 2015 (p < 0.001), mainly due to early decreases in the proportions of serotypes 3 and 19A. In contrast, during the same period, PCV7 serotypes (11.9% in 2012-2015) and the serotypes exclusive of the 23-valent polysaccharide vaccine (26.0% in 2012-2015), remained relatively stable, while non-vaccine types increased from 27.0% in 2010 to 41.9% in 2015 (p

Although serogroup 6 was among the first to be recognized among Streptococcus pneumoniae, several new serotypes were identified since the introduction of pneumococcal conjugate vaccines (PCVs). A decrease of the 6B-2 variant among invasive pneumococcal disease (IPD), but not 6B-1, was noted post conjugate vaccine introduction, underpinned by a decrease of CC273 isolates. Serotype 6C was associated with adult IPD and increased in this age group representing two lineages (CC315 and CC395), while the same lineages expressed other serogroup 6 serotypes in children. Taken together, these findings suggest a potential cross-protection of PCVs against serotype 6C IPD among vaccinated children but not among adults. Serotype 6A became the most important serogroup 6 serotype in children but it decreased in adult IPD. No other serogroup 6 serotypes were detected, so available phenotypic or simple genotypic assays remain adequate for distinguishing serotypes within serogroup 6 isolates.

Trees, including minimum spanning trees (MSTs), are commonly used in phylogenetic studies. But, for the research community, it may be unclear that the presented tree is just a hypothesis, chosen from among many possible alternatives. In this scenario, it is important to quantify our confidence in both the trees and the branches/edges included in such trees. In this paper, we address this problem for MSTs by introducing a new edge betweenness metric for undirected and weighted graphs. This spanning edge betweenness metric is defined as the fraction of equivalent MSTs where a given edge is present. The metric provides a per edge statistic that is similar to that of the bootstrap approach frequently used in phylogenetics to support the grouping of taxa. We provide methods for the exact computation of this metric based on the well known Kirchhoff's matrix tree theorem. Moreover, we implement and make available a module for the PHYLOViZ software and evaluate the proposed metric concerning both effectiveness and computational performance. Analysis of trees generated using multilocus sequence typing data (MLST) and the goeBURST algorithm revealed that the space of possible MSTs in real data sets is extremely large. Selection of the edge to be represented using bootstrap could lead to unreliable results since alternative edges are present in the same fraction of equivalent MSTs. The choice of the MST to be presented, results from criteria implemented in the algorithm that must be based in biologically plausible models.

Resumo: O Estudo Viriato é um estudo nacional, prospectivo e multicêntrico, de vigilância da susceptibilidade aos antimicrobianos de bactérias frequentemente responsáveis por infecções do aparelho respiratório adquiridas na comunidade. Nos anos de 2003 e 2004 participaram 29 laboratórios de todo o país. Isolaram-se 2945 microrganismos que foram estudados num laboratório coordenador. Das 513 estirpes de Streptococcus pyogenes de doentes com amigdalo-faringite aguda, todas eram susceptíveis à penicilina e outros antibióticos beta-lactâmicos, mas 18,9% eram resistentes à eritromicina, claritromicina e azitromicina. Nas estirpes resistentes foi mais frequente o fenótipo M (67,0%) que confere resistência à eritromicina (CIM90=16 mg/L), claritromicina e azitromicina, mas susceptibilidade à clindamicina (CIM90=0,094 mg/L). De doentes com infecção do aparelho respiratório inferior estudaram-se 1300 estirpes de Streptococcus pneumoniae (pneumococos), 829 de Haemophilus influenzae e 303 de Moraxella catarrhalis. Em S. pneumoniae, 18,4% das estirpes eram resistentes à penicilina (3,5% com resistência elevada), 7,1% à cefuroxima, 0,5% à amoxicilina, 0,5% à amoxicilina/clavulanato, 18,8% à eritromicina, claritromicina e azitromicina, 14,5 % à tetraciclina, 16,5% ao cotrimoxazol e 0,4% à levofloxacina. Nas estirpes resistentes aos macrólidos, dominou o fenótipo MLSB (83,7%), caracterizado por resistência elevada (CIM90 >256 mg/L) à eritromicina, claritromicina, azitromicina e clindamicina. Produziam beta-lactamase 10,0% de H. influenzae e 96,4% de M. catarrhalis. Em H. influenzae demonstrou-se 5,5% de resistência à claritromicina e 13,4% ao cotrimoxazol. A quase totalidade das estirpes era susceptível à amoxicilina / clavulanato, cefuroxima, azitromicina, tetraciclina e ciprofloxacina. Em M. catarrhalis a resistência ao co-trimoxazol foi de 27,1% e à tetraciclina de 1,0%. Todas as estirpes eram susceptíveis à amoxicilina / clavulanato, cefuroxima, claritromicina, azitromicina e ciprofloxacina. De entre o conjunto de antibióticos ensaiado, a penicilina continua a ser o mais activo contra S. pyogenes e a amoxicilina / clavulanato e as quinolonas os mais activos simultaneamente contra S. pneumoniae, H. influenzae e M. catarrhalis.Rev Port Pneumol 2006; XII (1): 9-29 Abstract: The Viriato Study is a nationwide, prospective, multicenter surveillance study of the antimicrobial susceptibility of bacterial pathogens commonly associated with community-acquired respiratory tract infections in Portugal. In 2003 and 2004 a total of 2945 isolates was recovered in the 29 laboratories that participated in the study. Testing was undertaken in a central laboratory. Of the 513 Streptococcus pyogenes strains isolated from patients with acute tonsillitis all were susceptible to penicillin and other beta-lactams but 18.9% were resistant to erythromycin, clarithromycin and azithromycin. The M phenotype dominated (67%), conferring resistance to erythromycin (MIC90=16 mg/L), clarythromycin and azithromycin, but susceptibility to clindamycin (MIC90=0.094 mg/L). From patients with lower respiratory tract infection 1,300 strains of Streptococcus pneumoniae, 829 of Haemophilus influenzae, and 303 of Moraxella catarrhalis were studied. Among S. pneumoniae isolates 18.4% were resistant to penicillin (3.5% showing high-level resistance), 7.1% to cefuroxime, 0.5% to amoxicillin and amoxicillin/ clavulanate, 18.8% to erythromycin, clarithromycin and azithromycin, 14.9% to tetracycline, 16.5% to co-trimoxazol, and 0.4% to levofloxacin. Beta-lactamases were produced by 10.0% of H. influenzae and 96.4% of M. catarrhalis. In H. influenzae resistance to clarithromycin was 5.5% and to cotrimoxazole was 13.4%. Most strains were susceptible to amoxicillin/clavulanate, cefuroxime, azithromycin, tetracycline and ciprofloxacin. In M. catarrhalis resistance to co-trimoxazole was 27.1% and to tetracycline 1.0%. All strains were susceptible to amoxicillin/clavulanate, cefuroxime, clarithromycin, azithromycin and ciprofloxacin. Penicillin was the most active antimicrobial agent against S. pyogenes and amoxycillin / clavulanate and the quinolones the most active in vitro simultaneously against S. pneumoniae, H. influenza and M. catarrhalis.Rev Port Pneumol 2006; XII (1): 9-29 Palavras-chave: Portugal, Estudo Viriato, infecção respiratória, comunidade, 2003, 2004, susceptibilidade aos antimicrobianos, Streptococcus pyogenes, Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, Key-words: Portugal, Viriato Study, respiratory tract infections, community, 2003, 2004, antimicrobial resistance, Streptococcus pyogenes, Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis

In Streptococcus pneumoniae the competence-stimulating peptide (CSP), encoded by the comC gene, controls competence development and influences biofilm growth. We explored the influence of pherotype, defined by the two major comC allelic variants (comC1 and comC2), on biofilm development and recombination efficiency. Among isolates recovered from human infections those presenting comC1 show a higher capacity to form in vitro biofilms. The influence of pherotype on biofilm growth was confirmed by experiments with isogenic strains differing in their comC alleles. Biofilm architecture evaluated by confocal laser scanning microscopy showed that strains carrying comC1 form biofilms that are denser and thicker than those carrying the comC2 allele. Isogenic strains carrying the comC1 allele yielded more transformants than those carrying the comC2 allele in both planktonic and biofilm growth. Transformation assays with comC knockout strains show that ComD1 needs lower doses of the signaling peptide to reach the same biological outcomes. In contrast to mixed planktonic growth, within mixed biofilms inter-pherotype genetic exchange is less frequent than that occurring between bacteria of the same pherotype. Since biofilms are a major bacterial lifestyle, these observations may explain the genetic differentiation between populations with different pherotypes reported previously. Considering that biofilms have been associated with colonization our results suggest that strains carrying the comC1 allele may be more transmissible and more efficient at persisting in carriage. Both effects may help explain the higher prevalence of the comC1 allele in the pneumococcal population.

There is limited information on the serotypes causing non-invasive pneumococcal pneumonia (NIPP). Our aim was to characterize pneumococci causing NIPP in adults to determine recent changes in serotype prevalence, the potential coverage of pneumococcal vaccines and changes in antimicrobial resistance. Serotypes and antimicrobial susceptibility profiles of a sample of 1300 isolates recovered from adult patients (≥18 yrs) between 1999 and 2011 (13 years) were determined. Serotype 3 was the most frequent cause of NIPP accounting for 18% of the isolates. The other most common serotypes were 11A (7%), 19F (7%), 19A (5%), 14 (4%), 22F (4%), 23F (4%) and 9N (4%). Between 1999 and 2011, there were significant changes in the proportion of isolates expressing vaccine serotypes, with a steady decline of the serotypes included in the 7-valent conjugate vaccine from 31% (1999-2003) to 11% (2011) (P

This protocol was developed in a study aimed to determine the cellular localization of the lysin of pneumococcal phage SV1 (Frias et al., 2013). We obtained proteins from the surface of Streptococcus pneumoniae by elution with choline or those secreted to the medium. The analysis by Western blot of these fractions allowed us to demonstrate that the phage lysin localizes to the cell wall, associating with choline residues in the teichoic acids. Hence, protein extracts can be used to determine the localization of uncharacterized proteins and can also be useful for other biochemical analyses such as protein identification. This protocol can be easily adapted to different pneumococcal strains and growth conditions and it is well suited to isolate other proteins of interest.

In Portugal, pneumococcal conjugate vaccines have been administered to children outside of the national immunization plan since 2001. We determined the serotype and antimicrobial susceptibility of 1265 isolates responsible for adult invasive pneumococcal infections (IPD) between 2009 and 2011 and compared the results with previously published data from 1999 to 2008. Serotypes 3 (12.6%), 7F (10.0%), 19A (9.1%), 14 (8.4%), 1 (6.9%) and 8 (6.2%) were the most frequent and together accounted for 53.2% of adult IPD. Serotypes 1 and 5 declined significantly while serotype 34, not included in any vaccine, increased. Taken together, the serotypes included in the 13-valent conjugate vaccine (PCV13) peaked among adult IPD isolates in 2008 (70.2%) and declined since then reaching 53.5% in 2011. The decline in the serotypes included in the 23-valent polysaccharide vaccine since 2007 was also significant but much more modest with 79.2% of the isolates causing IPD in 2011 expressing these serotypes. Since the changes in serotypes causing IPD in adults coincided with the 10-valent and PCV13 introduction in children, it is unlikely that vaccination triggered these changes although it may have accelerated them. The proportion of IPD caused by serotypes included in the 7-valent conjugate vaccine remained stable (19.0%). Both penicillin non-susceptibility and erythromycin resistance increased in the study period, with serotypes 14 and 19A accounting for the majority of resistant isolates.

Several research fields frequently deal with the analysis of diverse classification results of the same entities. This should imply an objective detection of overlaps and divergences between the formed clusters. The congruence between classifications can be quantified by clustering agreement measures, including pairwise agreement measures. Several measures have been proposed and the importance of obtaining confidence intervals for the point estimate in the comparison of these measures has been highlighted. A broad range of methods can be used for the estimation of confidence intervals. However, evidence is lacking about what are the appropriate methods for the calculation of confidence intervals for most clustering agreement measures. Here we evaluate the resampling techniques of bootstrap and jackknife for the calculation of the confidence intervals for clustering agreement measures. Contrary to what has been shown for some statistics, simulations showed that the jackknife performs better than the bootstrap at accurately estimating confidence intervals for pairwise agreement measures, especially when the agreement between partitions is low. The coverage of the jackknife confidence interval is robust to changes in cluster number and cluster size distribution.

A resistência aos antimicrobianos em bactérias de Gram--positivo é um problema de importância crescente. As espécies Streptococcus pneumoniae, Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecium e Enterococcus faecalis apresentam-se cada vez mais resistentes aos antimicrobianos de primeira linha no tratamento das infecções em que são agentes etiológicos. Far-se-á uma revisão dos principais mecanismos de resistência existentes em cada uma das espécies e da sua disseminação na população bacteriana. Será discutida a evolução da proporção de estirpes resistentes em Portugal desde 1993 até 2008 e qual o seu enquadramento no contexto europeu. Discutir-se-ão as alternativas terapêuticas que mais recentemente chegaram ao mercado e qual a situação actual de resistência a estes novos fármacos.

Streptococcus dysgalactiae subspecies equisimilis (SDSE) is an emerging global pathogen that can colonize and infect humans. Although most SDSE isolates possess the Lancefield group G carbohydrate, a significant minority have the group C carbohydrate. Isolates are further sub-typed on the basis of differences within the emm gene. To gain a better understanding of their molecular epidemiology and evolutionary relationships, multilocus sequence typing (MLST) analysis was performed on SDSE isolates collected from Australia, Europe and North America.The 178 SDSE isolates, representing 37 emm types, segregate into 80 distinct sequence types (STs) that form 17 clonal complexes (CCs). Eight STs recovered from all three continents account for >50% of the isolates. Thus, a small number of STs are highly prevalent and have a wide geographic distribution. Both ST and CC strongly correlate with group carbohydrate. In contrast, eleven STs were associated with >1 emm type, suggestive of recombinational replacements involving the emm gene; furthermore, 35% of the emm types are associated with genetically distant STs. Data also reveal a history of extensive inter- and intra-species recombination involving the housekeeping genes used for MLST. Sequence analysis of single locus variants identified through goeBURST indicates that genetic change mediated by recombination occurred approximately 4.4 times more frequently than by point mutation.A few genetic lineages with an intercontinental distribution dominate among SDSE causing infections in humans. The distinction between group C and G isolates reflects recent evolution, and no long-term genetic isolation between them was found. Lateral gene transfer and recombination involving housekeeping genes and the emm gene are important mechanisms driving genetic variability in the SDSE population.

Streptococcus pneumoniae (pneumococcus) is able to form biofilms in vivo and previous studies propose that pneumococcal biofilms play a relevant role both in colonization and infection. Additionally, pneumococci recovered from human infections are characterized by a high prevalence of lysogenic bacteriophages (phages) residing quiescently in their host chromosome. We investigated a possible link between lysogeny and biofilm formation. Considering that extracellular DNA (eDNA) is a key factor in the biofilm matrix, we reasoned that prophage spontaneous activation with the consequent bacterial host lysis could provide a source of eDNA, enhancing pneumococcal biofilm development. Monitoring biofilm growth of lysogenic and non-lysogenic pneumococcal strains indicated that phage-infected bacteria are more proficient at forming biofilms, that is their biofilms are characterized by a higher biomass and cell viability. The presence of phage particles throughout the lysogenic strains biofilm development implicated prophage spontaneous induction in this effect. Analysis of lysogens deficient for phage lysin and the bacterial major autolysin revealed that the absence of either lytic activity impaired biofilm development and the addition of DNA restored the ability of mutant strains to form robust biofilms. These findings establish that limited phage-mediated host lysis of a fraction of the bacterial population, due to spontaneous phage induction, constitutes an important source of eDNA for the S. pneumoniae biofilm matrix and that this localized release of eDNA favors biofilm formation by the remaining bacterial population.

Very diverse research fields frequently deal with the analysis of multiple clustering results, which should imply an objective detection of overlaps and divergences between the formed groupings. The congruence between these multiple results can be quantified by clustering comparison measures such as the Wallace coefficient (W). Since the measured congruence is dependent on the particular sample taken from the population, there is variability in the estimated values relatively to those of the true population. In the present work we propose the use of a confidence interval (CI) to account for this variability when W is used. The CI analytical formula is derived assuming a Gaussian sampling distribution and recurring to the algebraic relationship between W and the Simpson's index of diversity. This relationship also allows the estimation of the expected Wallace value under the assumption of independence of classifications. We evaluated the CI performance using simulated and published microbial typing data sets. The simulations showed that the CI has the desired 95% coverage when the W is greater than 0.5. This behaviour is robust to changes in cluster number, cluster size distributions and sample size. The analysis of the published data sets demonstrated the usefulness of the new CI by objectively validating some of the previous interpretations, while showing that other conclusions lacked statistical support.

Highlights: • La combinación de diversos métodos digitales para la reconstrucción virtual es una poderosa herramienta para la difusión y la investigación del patrimonio arqueológico. • El trabajo con cientos de fragmentos hallados en la tumba de Henenu ha permitido la reconstrucción virtual de cuatro estelas funerarias, con especial detalle en el caso de la estela A. • Las hipótesis, cálculos e interpretaciones derivadas de estas reconstrucciones virtuales han contribuido a identificar la posición inicial de estas estelas en el monumento. Resumen: Este trabajo pretende demostrar cómo la combinación de métodos digitales para la reconstrucción virtual en arqueología es mucho más que una simple herramienta para la difusión del patrimonio. A través del estudio de la tumba de Henenu (TT 313: Deir el-Bahari, Luxor), este artículo demuestra que los métodos de reconstrucción virtual constituyen una herramienta eficaz para examinar diversas hipótesis. Cientos de fragmentos de estelas fueron encontrados por la expedición del Museo Metropolitano (Nueva York) en 1922-1923. Tras este descubrimiento, los arqueólogos reconstruyeron cuatro estelas, destacando la estela A. Una vez recuperados los datos arqueológicos y arquitectónicos, los especialistas del MKTP procedieron a reconstruir las posibles dimensiones de estas estelas mediante técnicas digitales. Además, se examinó si las estelas encajaban en los nichos originales construidos para este propósito. En este artículo se plantea el flujo de trabajo empleado desde el escaneado láser hasta el modelado de las estelas, usando un escáner láser para documentar la tumba. Los datos fueron procesados con Leica Cyclone para obtener una malla, aunque la malla presentaba algo de “ruido”, anomalía que debió ser corregida con otro software. El resultado fue entonces exportado a Blender. De este modo se consiguió la reconstrucción virtual de la estela A de Henenu, que recibió texturas y otros rasgos con Substance Painter y Photoshop. El tema principal de la estela es la provisión de ofrendas y la garantía de beneficios para el difunto, lo que explicaría que el nicho de erección de la estela estuviera a la entrada de la tumba. Como se describe en las conclusiones, gracias a la aplicación de las técnicas de reconstrucción virtual y a los cálculos derivados de la misma, se ha podido elegir una de las hipótesis de reconstrucción y localización de las estelas, ofreciendo una respuesta a la pregunta inicial que se planteaba sobre el caso de la estela A.

Whole genome sequencing (WGS) has become a vital tool in clinical microbiology, playing an important role in outbreak investigations, molecular surveillance, and identification of bacterial species, resistance mechanisms and virulence factors. However, the complexity of WGS data presents challenges in interpretation and reporting, requiring tailored strategies to enhance efficiency and impact. This study explores the diverse needs of key stakeholders in healthcare, including clinical management, laboratory work, public surveillance and epidemiology, infection prevention and control, and academic research, regarding WGS-based reporting of clinically relevant bacterial species. In order to determine preferences regarding WGS reports, human-centered design approach was employed, involving an online survey and a subsequent workshop with stakeholders. The survey gathered responses from 64 participants representing the above mentioned healthcare sectors across geographical regions. Key findings include the identification of barriers related to data accessibility, integration with patient records, and the complexity of interpreting WGS results. As the participants designed their ideal report using nine pre-defined sections of a typical WGS report, differences in needs regarding report structure and content across stakeholders became evident. The workshop discussions further highlighted the need to feature critical findings and quality metrics prominently in reports, as well as the demand for flexible report designs. Commonalities were observed across stakeholder-specific reporting templates, such as the uniform ranking of certain report sections, but preferences regarding the depth of content within these sections varied. Using these findings, we suggest stakeholder-specific structures which should be considered when designing customized reporting templates. In conclusion, this study underscores the importance of tailoring WGS-based reports of clinically relevant bacteria to meet the distinct needs of diverse healthcare stakeholders. The evolving landscape of digital reporting increases the opportunities with respect to WGS reporting and its utility in managing infectious diseases and public health surveillance.

Abstract Streptococcus dysgalactiae subsp. equisimilis (SDSE) is an emerging cause of human infection with invasive disease incidence and clinical manifestations comparable to the closely related species, Streptococcus pyogenes. Through systematic genomic analyses of 501 disseminated SDSE strains, we demonstrate extensive overlap between the genomes of SDSE and S. pyogenes. More than 75% of core genes are shared between the two species with one third demonstrating evidence of cross-species recombination. Twenty-five percent of mobile genetic element (MGE) clusters and 16 of 55 SDSE MGE insertion regions were shared across species. Assessing potential cross-protection from leading S. pyogenes vaccine candidates on SDSE, 12/34 preclinical vaccine antigen genes were shown to be present in >99% of isolates of both species. Relevant to possible vaccine evasion, six vaccine candidate genes demonstrated evidence of inter-species recombination. These findings demonstrate previously unappreciated levels of genomic overlap between these closely related pathogens with implications for streptococcal pathobiology, disease surveillance and prevention.