Comparison of next generation technologies and bioinformatics pipelines for capsular typing of Streptococcus pneumoniae.

Whole genome sequencing (WGS)-based approaches for pneumococcal capsular typing have become an alternative to serological methods. In silico serotyping from WGS has not yet been applied to long-read sequences produced by third-generation technologies. The objective of the study was to determine the capsular types of pneumococci causing invasive disease in Catalonia (Spain) using serological typing and WGS and to compare the performance of different bioinformatics pipelines using short- and long-read data from WGS. All invasive pneumococcal pediatric isolates collected in Hospital Sant Joan de Déu (Barcelona) from 2013 to 2019 were included. Isolates were assigned a capsular type by serological testing based on anticapsular antisera and by different WGS-based pipelines: Illumina sequencing followed by serotyping with PneumoCaT, SeroBA, and Pathogenwatch vs MinION-ONT sequencing coupled with serotyping by Pathogenwatch from pneumococcal assembled genomes. A total of 119 out of 121 pneumococcal isolates were available for sequencing. Twenty-nine different serotypes were identified by serological typing, with 24F (n = 17; 14.3%), 14 (n = 10; 8.4%), and 15B/C (n = 8; 6.7%) being the most common serotypes. WGS-based pipelines showed initial concordance with serological typing (>91% of accuracy). The main discrepant results were found at the serotype level within a serogroup: 6A/B, 6C/D, 9A/V, 11A/D, and 18B/C. Only one discrepancy at the serogroup level was observed: serotype 29 by serological testing and serotype 35B/D by all WGS-based pipelines. Thus, bioinformatics WGS-based pipelines, including those using third-generation sequencing, are useful for pneumococcal capsular assignment. Possible discrepancies between serological typing and WGS-based approaches should be considered in pneumococcal capsular-type surveillance studies.

Dynamics of Pneumococcal Carriage in Adults: A New Look at an Old Paradigm.

BACKGROUND: Limited information is available on pneumococcal colonization among adults. We studied pneumococcal carriage dynamics in healthy adults using high-sensitivity approaches. METHODS: Eighty-seven adults (25-50 years old) were followed for 6 months in Portugal. Nasopharyngeal, oropharyngeal, and saliva samples were obtained monthly; pneumococcal carriers were also sampled weekly. Carriage was investigated by quantitative polymerase chain reaction (targeting lytA and piaB) and culture. Positive samples were serotyped. RESULTS: Approximately 20% of the adults were intermittent carriers; 10% were persistent carriers (>4 months). Pneumococcal acquisition and clearance rates were 16.5 (95% confidence interval [CI], 11.2-24.2) and 95.9 (95% CI, 62.3-145.0) cases/1000 person-weeks, respectively. Living with children increased pneumococcal acquisition (hazard ratio, 9.7 [95% CI, 2.6-20.5]; P < .001). Median duration of carriage was 7 weeks and did not depend on regular contact with children. CONCLUSIONS: The pneumococcal carrier state in healthy adults is more dynamic than generally assumed: Acquisition is frequent and duration of carriage is often long. This suggests that some adults may act as reservoirs of pneumococci and hence, depending on the social structure of a community, the magnitude of herd effects potentially attainable through children vaccination may vary. These findings are important when designing strategies to prevent pneumococcal disease in adults.

Carriage of multiple Streptococcus pneumoniae capsular types is frequent among children with invasive pneumococcal disease.

Streptococcus pneumoniae (pneumococcus) is a human pathogen that colonizes the nasopharynx. We investigated serotype distribution in paired invasive and nasopharyngeal samples obtained from 57 children during invasive pneumococcal disease. Of 39 nasopharyngeal samples positive for pneumococci, 46.2% contained a serotype different from the one causing disease. This study reports a high frequency of pneumococcal multiple serotype carriage in children with invasive pneumococcal disease. Whether multiple serotype carriage is important for the onset and progress to pneumococcal infection warrants further investigation.

The blp Locus of Streptococcus pneumoniae Plays a Limited Role in the Selection of Strains That Can Cocolonize the Human Nasopharynx.

UNLABELLED: Nasopharyngeal colonization is important for Streptococcus pneumoniae evolution, providing the opportunity for horizontal gene transfer when multiple strains co-occur. Although colonization with more than one strain of pneumococcus is common, the factors that influence the ability of strains to coexist are not known. A highly variable blp (bacteriocin-like peptide) locus has been identified in all sequenced strains of S. pneumoniae This locus controls the regulation and secretion of bacteriocins, small peptides that target other bacteria. In this study, we analyzed a series of cocolonizing isolates to evaluate the impact of the blp locus on human colonization to determine whether competitive phenotypes of bacteriocin secretion restrict cocolonization. We identified a collection of 135 nasopharyngeal samples cocolonized with two or more strains, totaling 285 isolates. The blp locus of all strains was characterized genetically with regard to pheromone type, bacteriocin/immunity content, and potential for locus functionality. Inhibitory phenotypes of bacteriocin secretion and locus activity were assessed through overlay assays. Isolates from single colonizations (n = 298) were characterized for comparison. Cocolonizing strains had a high diversity of blp cassettes; approximately one-third displayed an inhibitory phenotype in vitro Despite in vitro evidence of competition, pneumococci cocolonized the subjects independently of blp pheromone type (P = 0.577), bacteriocin/immunity content, blp locus activity (P = 0.798), and inhibitory phenotype (P = 0.716). In addition, no significant differences were observed when single and cocolonizing strains were compared. Despite clear evidence of blp-mediated competition in experimental models, the results of our study suggest that the blp locus plays a limited role in restricting pneumococcal cocolonization in humans. IMPORTANCE: Nasopharyngeal colonization with Streptococcus pneumoniae (pneumococcus) is important for pneumococcal evolution, as the nasopharynx represents the major site for horizontal gene transfer when multiple strains co-occur, a phenomenon known as cocolonization. Understanding how pneumococcal strains interact within the competitive environment of the nasopharynx is of chief importance in the context of pneumococcal ecology. In this study, we used an unbiased collection of naturally co-occurring pneumococcal strains and showed that a biological process frequently used by bacteria for competition-bacteriocin production-is not decisive in the coexistence of pneumococci in the host, in contrast to what has been shown in experimental models.

How direct competition shapes coexistence and vaccine effects in multi-strain pathogen systems.

We describe an integrated modeling framework for understanding strain coexistence in polymorphic pathogen systems. Previous studies have debated the utility of neutral formulations and focused on cross-immunity between strains as a major stabilizing mechanism. Here we convey that direct competition for colonization mediates stable coexistence only when competitive abilities amongst pathogen clones satisfy certain pairwise asymmetries. We illustrate our ideas with nested SIS models of single and dual colonization, applied to polymorphic pneumococcal bacteria. By fitting the models to cross-sectional prevalence data from Portugal (before and after the introduction of a seven-valent pneumococcal conjugate vaccine), we are able to not only statistically compare neutral and non-neutral epidemiological formulations, but also estimate vaccine efficacy, transmission and competition parameters simultaneously. Our study highlights that the response of polymorphic pathogen populations to interventions holds crucial information about strain interactions, which can be extracted by suitable nested modeling.

Variable recombination dynamics during the emergence, transmission and 'disarming' of a multidrug-resistant pneumococcal clone.

BACKGROUND: Pneumococcal ß-lactam resistance was first detected in Iceland in the late 1980s, and subsequently peaked at almost 25% of clinical isolates in the mid-1990s largely due to the spread of the internationally-disseminated multidrug-resistant PMEN2 (or Spain6B-2) clone of Streptococcus pneumoniae. RESULTS: Whole genome sequencing of an international collection of 189 isolates estimated that PMEN2 emerged around the late 1960s, developing resistance through multiple homologous recombinations and the acquisition of a Tn5253-type integrative and conjugative element (ICE). Two distinct clades entered Iceland in the 1980s, one of which had acquired a macrolide resistance cassette and was estimated to have risen sharply in its prevalence by coalescent analysis. Transmission within the island appeared to mainly emanate from Reykjavík and the Southern Peninsular, with evolution of the bacteria effectively clonal, mainly due to a prophage disrupting a gene necessary for genetic transformation in many isolates. A subsequent decline in PMEN2's prevalence in Iceland coincided with a nationwide campaign that reduced dispensing of antibiotics to children in an attempt to limit its spread. Specific mutations causing inactivation or loss of ICE-borne resistance genes were identified from the genome sequences of isolates that reverted to drug susceptible phenotypes around this time. Phylogenetic analysis revealed some of these occurred on multiple occasions in parallel, suggesting they may have been at least temporarily advantageous. However, alteration of 'core' sequences associated with resistance was precluded by the absence of any substantial homologous recombination events. CONCLUSIONS: PMEN2's clonal evolution was successful over the short-term in a limited geographical region, but its inability to alter major antigens or 'core' gene sequences associated with resistance may have prevented persistence over longer timespans.

Non-typeable pneumococci circulating in Portugal are of cps type NCC2 and have genomic features typical of encapsulated isolates.

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.

A one-step low-cost molecular test for SARS-CoV-2 detection suitable for community testing using minimally processed saliva.

The gold standard for coronavirus disease 2019 diagnostic testing relies on RNA extraction from naso/oropharyngeal swab followed by amplification through reverse transcription-polymerase chain reaction (RT-PCR) with fluorogenic probes. While the test is extremely sensitive and specific, its high cost and the potential discomfort associated with specimen collection made it suboptimal for public health screening purposes. In this study, we developed an equally reliable, but cheaper and less invasive alternative test based on a one-step RT-PCR with the DNA-intercalating dye SYBR Green, which enables the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) directly from saliva samples or RNA isolated from nasopharyngeal (NP) swabs. Importantly, we found that this type of testing can be fine-tuned to discriminate SARS-CoV-2 variants of concern. The saliva RT-PCR SYBR Green test was successfully used in a mass-screening initiative targeting nearly 4500 asymptomatic children under the age of 12. Testing was performed at a reasonable cost, and in some cases, the saliva test outperformed NP rapid antigen tests in identifying infected children. Whole genome sequencing revealed that the antigen testing failure could not be attributed to a specific lineage of SARS-CoV-2. Overall, this work strongly supports the view that RT-PCR saliva tests based on DNA-intercalating dyes represent a powerful strategy for community screening of SARS-CoV-2. The tests can be easily applied to other infectious agents and, therefore, constitute a powerful resource for an effective response to future pandemics.

Comparative analysis of Streptococcus pneumoniae transmission in Portuguese and Finnish day-care centres.

BACKGROUND: Day-care centre (DCC) attendees play a central role in maintaining the circulation of Streptococcus pneumoniae (pneumococcus) in the population. The prevalence of pneumococcal carriage is highest in DCC attendees but varies across countries and is found to be consistently lower in Finland than in Portugal. We compared key parameters underlying pneumococcal transmission in DCCs to understand which of these contributed to the observed differences in carriage prevalence. METHODS: Longitudinal data about serotype-specific carriage in DCC attendees in Portugal (47 children in three rooms; mean age 2 years; range 1-3 years) and Finland (91 children in seven rooms; mean age 4 years; range 1-7 years) were analysed with a continuous-time event history model in a Bayesian framework. The monthly rates of within-room transmission, community acquisition and clearing carriage were estimated. RESULTS: The posterior mean of within-room transmission rate was 1.05 per month (Portugal) vs. 0.63 per month (Finland). The smaller rate of clearance in Portugal (0.57 vs. 0.73 per month) is in accordance with the children being younger. The overall community rate of acquisition was larger in the Portuguese setting (0.25 vs. 0.11 per month), in agreement with that the groups belonged to a larger DCC. The model adequately predicted the observed levels of carriage prevalence and longitudinal patterns in carriage acquisition and clearance. CONCLUSIONS: The difference in prevalence of carriage (61% in Portuguese vs. 26% among Finnish DCC attendees) was assigned to the longer duration of carriage in younger attendees and a significantly higher rate of within-room transmission and community acquisition in the Portuguese setting.

Clonal Changes in the Pneumococcal Population Carried by Portuguese Children during Six Years of Private Use of the 13-Valent Pneumococcal Conjugate Vaccine: the Relative Contribution of Clonal Expansion, Clonal Emergence, and Capsular Switch Events.

In Portugal, the 13-valent pneumococcal conjugate vaccine (PCV13) was available for private use from 2010 to 2015 and it was introduced in the National Immunization Program in 2015. We have reported that private use of PCV13 led to extensive serotype replacement and an increase in antimicrobial susceptibility among pneumococci carried by healthy children. We investigated which clonal changes concurred with these observations. A total of 657 pneumococcal strains, representative of a collection of 2,615 isolates, were genotyped by multilocus sequence typing (MLST). The isolates were recovered in 2009 to 2010 (pre-PCV13), 2011 to 2012 (early PCV13), and 2015 to 2016 (late PCV13) from children attending day care centers in two regions of Portugal (one urban, one rural). One-hundred seventy-one sequence types (STs) were identified, corresponding to 18 clonal complexes (CCs) and 58 singletons. Most CCs (n = 17) and several singletons (n = 16) were found in both regions, indicating that they were geographically disseminated. Clonal complexes expressing PCV13 serotypes in circulation in the late PCV13 period were a subset of the ones identified in the pre-PCV13 period and were often associated with antimicrobial resistance. Among those, the most frequent in both regions was CC179, a multidrug-resistant clone of serotype 19F. Serotype replacement, following PCV13 use, was mainly due to expansion of the susceptible lineages expressing non-PCV13 serotypes already in circulation in the pre-PCV13 period. The emergence of ST53, associated with serotype 8, a major cause of disease in several European countries, was observed in the rural region. Potential capsular switching events, unrelated to PCV13 use, were detected. This study improves our understanding of changes triggered by the private use of PCV13 in Portugal. IMPORTANCE Streptococcus pneumoniae (pneumococcus) is a major human respiratory pathogen linked with high morbidity, mortality, and health care-associated costs worldwide. This bacterium often colonizes asymptomatically healthy children. Colonization is a prerequisite for disease and is also essential for transmission between individuals. The 13-valent pneumococcal conjugate vaccine targets 13 of 101 capsular types of pneumococci described to date. This vaccine not only prevents pneumococcal disease but also impacts colonization by decreasing the carriage of vaccine serotypes. Consequently, serotype replacement occurs. The clonal changes occurring during serotype replacement may be due to various mechanisms, such as clonal expansion, emergence, extinction, or capsular switch (vaccine escape). This study shows that in Portugal, the use of PCV13 has led to significant changes in clonal composition and that these were mainly due to the clonal expansion of lineages expressing serotypes not included in the vaccine.

Streptococcus pneumoniae carriage studies in adults: Importance, challenges, and key issues to consider when using quantitative PCR-based approaches.

Streptococcus pneumoniae causes significant morbidity and mortality among older adults. Detection of pneumococcal carriage is an accepted endpoint in pneumococcal conjugate vaccine studies. However, low sensitivity of culture-based approaches and nasopharyngeal samples have hampered adult S. pneumoniae carriage studies in the past. In contrast, detection of adult S. pneumoniae carriers with qPCR-based approaches can achieve high sensitivity and specificity and qPCR-based testing of oral samples improves accuracy of adult carriage detection. In this Viewpoint we outline a strategy for accurate qPCR-based testing. We recommend a dual-target approach for S. pneumoniae qPCR detection as no genetic target is universally present among or solely unique to it. Furthermore, we advise the evaluation of concordance among quantified qPCR targets to improve the accuracy of S. pneumoniae testing and qPCR-based serotyping. We do not recommend omission of qPCR-based oral sample testing as it will likely result in an underestimation of true adult carrier rates.

The upper respiratory tract microbiota of healthy adults is affected by Streptococcus pneumoniae carriage, smoking habits, and contact with children.

BACKGROUND: The microbiota of the upper respiratory tract is increasingly recognized as a gatekeeper of respiratory health. Despite this, the microbiota of healthy adults remains understudied. To address this gap, we investigated the composition of the nasopharyngeal and oropharyngeal microbiota of healthy adults, focusing on the effect of Streptococcus pneumoniae carriage, smoking habits, and contact with children. RESULTS: Differential abundance analysis indicated that the microbiota of the oropharynx was significantly different from that of the nasopharynx (P < 0.001) and highly discriminated by a balance between the classes Negativicutes and Bacilli (AUC of 0.979). Moreover, the oropharynx was associated with a more homogeneous microbiota across individuals, with just two vs. five clusters identified in the nasopharynx. We observed a shift in the nasopharyngeal microbiota of carriers vs. noncarriers with an increased relative abundance of Streptococcus, which summed up to 30% vs. 10% in noncarriers and was not mirrored in the oropharynx. The oropharyngeal microbiota of smokers had a lower diversity than the microbiota of nonsmokers, while no differences were observed in the nasopharyngeal microbiota. In particular, the microbiota of smokers, compared with nonsmokers, was enriched (on average 16-fold) in potential pathogenic taxa involved in periodontal diseases of the genera Bacillus and Burkholderia previously identified in metagenomic studies of cigarettes. The microbiota of adults with contact with children resembled the microbiota of children. Specifically, the nasopharyngeal microbiota of these adults had, on average, an eightfold increase in relative abundance in Streptococcus sp., Moraxella catarrhalis, and Haemophilus influenzae, pathobionts known to colonize the children's upper respiratory tract, and a fourfold decrease in Staphylococcus aureus and Staphylococcus lugdunensis. CONCLUSIONS: Our study showed that, in adults, the presence of S. pneumoniae in the nasopharynx is associated with a shift in the microbiota and dominance of the Streptococcus genus. Furthermore, we observed that smoking habits are associated with an increase in bacterial genera commonly linked to periodontal diseases. Interestingly, our research also revealed that adults who have regular contact with children have a microbiota enriched in pathobionts frequently carried by children. These findings collectively contribute to a deeper understanding of how various factors influence the upper respiratory tract microbiota in adults. Video Abstract.

Evidence for the intermediate disturbance hypothesis and exponential decay in replacement in Streptococcus pneumoniae following use of conjugate vaccines.

Understanding how pneumococci respond to pneumococcal conjugate vaccines (PCVs) is crucial to predict the impact of upcoming higher-valency vaccines. However, stages in pneumococcal community succession following disturbance are poorly understood as long-time series on carriage are scarce and mostly evaluated at end-point measurements. We used a 20-year cross-sectional dataset of pneumococci carried by Portuguese children, and methods from community ecology, to study community assembly and diversity following use of PCV7 and PCV13. Two successional stages were detected upon introduction of each PCV: one in which non-vaccine serotypes increased in abundance, fitted by a broken-stick model, and a second in which the community returned to the original structure, fitted by a geometric series, but with different serotype profile and a drop in richness as great as 24%. A peak in diversity was observed for levels of intermediate vaccine uptake (30-40%) in agreement with the intermediate disturbance hypothesis. Serotype replacement was fitted by an exponential decay model (R2 = 80%, P < 0.001). The half-life for replacement was 8 years for PCV7 and 10 years for PCV13. The structure of the pneumococcal community is resilient to vaccine pressure. The increasing loss of diversity, however, suggests it could eventually reach a threshold beyond which it may no longer recover.

Evaluation of Methicillin-Resistant Staphylococcus aureus Carriage in the Elderly in Portugal Using Selective Enrichment Followed by Quantitative Real-Time PCR.

The prevalence of methicillin-resistant Staphylococcus aureus (MRSA) in hospitals in Portugal is worrisome and among the highest in Europe. Surprisingly, MRSA prevalence in the community was described as very low (<2%) based on studies that used classical culture-based methods (CCBM). We investigated whether the apparent limited spread of MRSA in the community in Portugal might result from low sensitivity of CCBM. Nasopharyngeal- and oropharyngeal-paired samples obtained from senior adults living in nursing (n = 299) or family homes (n = 300) previously characterized by CCBM were reanalyzed. Samples were inoculated in a semi-selective enrichment medium, and those showing visible growth were evaluated by qPCR targeting nuc, mecA, and mecC genes (SSE+qPCR). By SSE+qPCR, 34 of the 1,198 (2.8%) samples were MRSA positive compared with 21 (1.8%) by CCBM. SSE+qPCR improved non-significantly detection of MRSA carriers from 5.4% to 8.0% (p = 0.12) in the nursing home collection, and from 0.3% to 1.7% (p = 0.13) in the family home collection. MRSA isolates belonged to three HA-MRSA clones widely disseminated in Portuguese hospitals. In conclusion, use of semi-selective medium combined with qPCR did not change the overall scenario previously described. In Portugal, MRSA circulation in the community among senior adults is low.

Haemophilus influenzae Carriage among Healthy Children in Portugal, 2015-2019.

Haemophilus influenzae is an important cause of mucosal and invasive infections and a common colonizer of the upper respiratory tract. As there are no recent data on H. influenzae carriage in Portugal, we aimed to characterize carriage samples and investigate possible parallelisms with disease isolates. Between 2016-2019, 1524 nasopharyngeal samples were obtained from children (0-6 years) attending day-care. H. influenzae were serotyped and screened for ß-lactamase production. Strains producing ß-lactamase and/or those that were encapsulated were further characterized by antibiotype; encapsulated strains were also investigated for MLST and the presence of antimicrobial resistance and virulence genes (extracted from whole genome sequencing). The overall carriage rate was 84.1%. Most isolates (96.7%) were nonencapsulated. Encapsulated strains were of serotypes f (1.8%), e (1.1%), a (0.3%), and b (0.1%). MLST showed clonality within serotypes. Although the lineages were the same as those that were described among disease isolates, colonization isolates had fewer virulence determinants. Overall, 7.5% of the isolates were ß-lactamase positive; one isolate had blaTEM-82, which has not been previously described in H. influenzae. A single isolate, which was identified as H. parainfluenzae, had an incomplete f-like cap locus. In conclusion, circulation of serotype b is residual. The few encapsulated strains are genetically related to disease-causing isolates. Thus, surveillance of H. influenzae carriage should be maintained.

Using Whole Genome Sequencing to Investigate a Mock-Outbreak of Carbapenem-Resistant Klebsiella pneumoniae in Real-Time.

INTRODUCTION: Healthcare associated infections due to carbapenem-resistant Klebsiella pneumoniae (CRKP) are a major concern in Portuguese hospitals. Whole genome sequencing (WGS) can improve infection control, but this practice is not routinely used by hospital clinical laboratories in Portugal. We simulated the investigation of a CRKP outbreak based on WGS, with the aim of determining, in the minimum possible time, genetic relatedness between CRKP clinical and environmental isolates. MATERIAL AND METHODS: Ten CRKP clinical isolates routinely obtained in the hospital laboratory were used. Forty environmental samples - from sinks and sink drains of ward rooms - were collected. Environmental samples were plated on selective media and presumptive CRKP colonies were isolated. Total DNA was extracted from all putative CRKP isolates and sequenced. Clonal relatedness was determined by multi-locus sequence typing and core genome single nucleotide polymorphism analysis; the presence of carbapenemase genes was evaluated. RESULTS: Clinical isolates were characterized in 48 hours: eight strains were confirmed as CRKP, of which six were of ST13 and carried blaKPC-3. Environmental samples results were obtained in six days: eight CRKP were isolated from which five were of ST13 and carried blaKPC-3. Clinical and environmental ST13 isolates were highly related: ten (of 11) isolates differed from each other in < 0.001% of 2 172 367 core nucleotides. DISCUSSION: WGS can be used as a high-resolution effective tool to investigate healthcare associated infections and track routes of dissemination in real-time. CONCLUSION: In Portugal, routine use of WGS to improve infection control could thrive through collaborative initiatives between hospitals and research institutes.

Introdução: As infeções associadas aos cuidados de saúde por Klebsiella pneumoniae resistente aos carbapenemos (CRKP) são uma preocupação nos hospitais portugueses. A sequenciação total do genoma [whole genome sequencing (WGS)] pode ajudar no controlo de infecção, mas esta prática não é comummente utilizada nos laboratórios clínicos hospitalares em Portugal. O objetivo deste estudo foi simular a investigação de um surto causado por CRKP, utilizando WGS. Pretendia-se testar a utilização desta técnica e determinar, no menor tempo possível, relações genéticas entre estirpes. Material e Métodos: Foram analisados dez isolados clínicos de CRKP. Foram obtidas quarenta amostras ambientais que foram inoculadas em meio seletivo para isolamento de colónias sugestivas de CRKP e depois sequenciado o DNA total dos isolados presumptivamente identificados como CRKP A relação clonal entre as estirpes foi determinada por multi-locus sequence typing e análise de single nucleotide polymorphisms no genoma core. Foi determinada a presença de genes de carbapenemases. Resultados: Os isolados clínicos foram caraterizados em 48 horas: oito isolados foram confirmados como CRKP. A maioria pertencia ao ST13 (n = 6) e possuía o gene blaKPC-3. As amostras ambientais foram caraterizadas em seis dias: foram isoladas oito CRKP, das quais cinco eram ST13 e continham o gene blaKPC-3. Os isolados ST13 clínicos e ambientais eram muito semelhantes entre si: dez dos 11 isolados diferiam entre si em menos de 0,001% dos 2 172 367 nucleótidos core analisados. Discussão: A sequenciação total do genoma pode ser usada como uma ferramenta útil para investigar infecções nosocomiais e rastrear cadeias de disseminação em tempo real. Conclusão: Em Portugal, o uso desta técnica em controlo de infecção pode ser implementado através de colaborações entre hospitais e institutos de investigação.

Clonal evolution leading to maintenance of antibiotic resistance rates among colonizing Pneumococci in the PCV7 era in Portugal.

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: Sweden(15A)ST63 (serotypes 15A and 19A) and Denmark(14)ST230 (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.

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.

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.

Intra-Species Interactions in Streptococcus pneumoniae Biofilms.

Streptococcus pneumoniae is a human pathogen responsible for high morbidity and mortality worldwide. Disease is incidental and is preceded by asymptomatic nasopharyngeal colonization in the form of biofilms. Simultaneous colonization by multiple pneumococcal strains is frequent but remains poorly characterized. Previous studies, using mostly laboratory strains, showed that pneumococcal strains can reciprocally affect each other's colonization ability. Here, we aimed at developing a strategy to investigate pneumococcal intra-species interactions occurring in biofilms. A 72h abiotic biofilm model mimicking long-term colonization was applied to study eight pneumococcal strains encompassing 6 capsular types and 7 multilocus sequence types. Strains were labeled with GFP or RFP, generating two fluorescent variants for each. Intra-species interactions were evaluated in dual-strain biofilms (1:1 ratio) using flow cytometry. Confocal microscopy was used to image representative biofilms. Twenty-eight dual-strain combinations were tested. Interactions of commensalism, competition, amensalism and neutralism were identified. The outcome of an interaction was independent of the capsular and sequence type of the strains involved. Confocal imaging of biofilms confirmed the positive, negative and neutral effects that pneumococci can exert on each other. In conclusion, we developed an experimental approach that successfully discriminates pneumococcal strains growing in mixed biofilms, which enables the identification of intra-species interactions. Several types of interactions occur among pneumococci. These observations are a starting point to study the mechanisms underlying those interactions.