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.

Impact of private use of the 13-valent pneumococcal conjugate vaccine (PCV13) on pneumococcal carriage among Portuguese children living in urban and rural regions.

In Portugal, the 13-valent pneumococcal conjugate vaccine (PCV13) was commercially available between 2010 and 2015, following a decade of private use of PCV7. We evaluated changes on serotype distribution and antimicrobial susceptibility of pneumococci carried by children living in two regions of Portugal (one urban and one rural). Three epidemiological periods were defined: pre-PCV13 (2009-2010), early-PCV13 (2011-2012), and late-PCV13 (2015-2016). Nasopharyngeal samples (n = 4,232) were obtained from children 0-6 years old attending day-care centers. Private use of PCVs was very high in both regions (>75%). Pneumococcal carriage remained stable and high over time (62.1%, 62.4% and 61.6% (p = 0.909) in the urban region; and 59.8%, 62.8%, 59.5% (p = 0.543) in the rural region). Carriage of PCV7 serotypes remained low (5.3%, 7.8% and 4.3% in the urban region; and 2.5%, 3.7% and 4.8% in the rural region). Carriage of PCV13 serotypes not targeted by PCV7 decreased in both the urban (16.4%, 7.3%, and 1.6%; p < 0.001) and rural regions (13.2%, 7.8%, and 1.9%; p < 0.001). This decline was mostly attributable to serotype 19A (14.1%, 4.4% and 1.3% in the urban region; and 11.1%, 3.6% and 0.8% in the rural region, both p < 0.001). Serotype 3 declined over time in the urban region (10.1%, 4.4%, 0.8%; p < 0.001) and had no obvious trend in the rural region (4.2%, 6.7%, 2.4%; p = 0.505). Serotype 6C decreased in both regions while serotypes 11D, 15A/B/C, 16F, 21, 22F, 23A/B, 24F, 35F, and NT were the most prevalent in the late-PCV13 period. Intermediate resistance to penicillin and non-susceptibility to erythromycin decreased significantly in both regions (19.5%, 13.3%, and 9.3%; and 25.4%, 25.9%, and 13.4%; both p < 0.001, respectively in the urban region; and 12.4%, 11.1%, and 2.8% (p < 0.001); and 15.3%, 14.7%, and 9.2% (p = 0.037), respectively, in the rural region). In conclusion, private use of PCV13 led to significant changes on the pneumococcal population carried by children in Portugal.

Genomic Characterization of the Emerging Pathogen Streptococcus pseudopneumoniae.

Streptococcus pseudopneumoniae is a close relative of the major human pathogen S. pneumoniae It is increasingly associated with lower-respiratory-tract infections (LRTI) and a high prevalence of antimicrobial resistance (AMR). S. pseudopneumoniae is difficult to identify using traditional typing methods due to similarities with S. pneumoniae and other members of the mitis group (SMG). Using whole-genome sequencing of LRTI isolates and a comparative genomic approach, we found that a large number of pneumococcal virulence and colonization genes are present in the core S. pseudopneumoniae genome. We also reveal an impressive number of novel surface-exposed proteins encoded by the genome of this species. In addition, we propose a new and entirely specific molecular marker useful for the identification of S. pseudopneumoniae Phylogenetic analyses of S. pseudopneumoniae show that specific clades are associated with allelic variants of core proteins. Resistance to tetracycline and macrolides, the two most common types of resistance, were found to be encoded by Tn916-like integrating conjugative elements and Mega-2. Overall, we found a tight association of genotypic determinants of AMR and phenotypic AMR with a specific lineage of S. pseudopneumoniae Taken together, our results shed light on the distribution in S. pseudopneumoniae of genes known to be important during invasive disease and colonization and provide insight into features that could contribute to virulence, colonization, and adaptation.IMPORTANCES. pseudopneumoniae is an overlooked pathogen emerging as the causative agent of lower-respiratory-tract infections and associated with chronic obstructive pulmonary disease (COPD) and exacerbation of COPD. However, much remains unknown on its clinical importance and epidemiology, mainly due to the lack of specific markers to distinguish it from S. pneumoniae Here, we provide a new molecular marker entirely specific for S. pseudopneumoniae and offer a comprehensive view of the virulence and colonization genes found in this species. Finally, our results pave the way for further studies aiming at understanding the pathogenesis and epidemiology of S. pseudopneumoniae.

Fighting antibiotic resistance in Portuguese hospitals: Understanding antibiotic prescription behaviours to better design antibiotic stewardship programmes.

OBJECTIVES: Since physicians play an important role in antibiotic usage, it is vital to understand their antibiotic-prescribing behaviour and knowledge on antimicrobial resistance in order to develop and implement effective antibiotic stewardship interventions. The aim of this study was to evaluate Portuguese physicians' knowledge and to understand prescription behaviours, difficulties and barriers in their antibiotic prescription process in order to promote better and well-adapted antibiotic stewardship policies. METHODS: This study was conducted in 2016 using a self-administered questionnaire to physicians in two tertiary public hospitals from two different regions in Portugal. RESULTS: Participating physicians [response rate 47.6% (30/63)] identified antibiotic resistance as a global problem; however, one-third did not recognise antibiotic resistance as a major problem on their own hospital. Factors that most influenced antibiotic prescription were 'microbiology laboratory results', 'patient clinical situation' and patient 'co-morbidities'. On the other hand, 'colleagues' opinion' and 'costs control' were considered as less determining factors. Regarding difficulties and bottlenecks in the antibiotic prescription process, participant physicians reported 'lack of (or delayed) microbiological results' and 'no access to antibiotic susceptibility patterns' as major barriers. 'Education and training' was considered the most effective intervention to improve antibiotic prescription. CONCLUSION: These results suggest that the design and implementation of antibiotic stewardship interventions should provide better data management and sharing tools between physicians and the microbiology laboratory, especially through the creation of antimicrobial prescribing guidelines according to hospital epidemiology, and easy access to hospital antibiotic susceptibility patterns and epidemiological data.

Co-Design of a Computer-Assisted Medical Decision Support System to Manage Antibiotic Prescription in an ICU Ward.

About 37 thousand people die per year in Europe due to infections by resistant bacteria. Fighting antimicrobial resistances (AR) is a top priority to save lives and reduce costs. AR is triggered mostly by uncritical antibiotic prescription. This paper presents HAITool, a decision-making information system to support antibiotic prescription. The system was co-developed together with health professionals using Design Science Research Methodology, empowered with innovative data visualization techniques to improve AR management. HAITool includes integrated visualizations of patient, microbiology, and pharmacy data, facilitating clinical decision support, antibiotic prescriptions quality and antibiotic-resistant bacteria monitoring. It also includes an alert module that monitors conformance of antibiotic prescriptions with norms and guidelines. HAITool is evaluated using both the Österle principles and interviews with physicians and infection control team from three participant hospitals.

Prevention and Control of Antimicrobial Resistant Healthcare-Associated Infections: The Microbiology Laboratory Rocks!

In Europe, each year, more than four milion patients acquire a healthcare-associated infection (HAI) and almost 40 thousand die as a direct consequence of it. Regardless of many stategies to prevent and control HAIs, they remain an important cause of morbidity and mortality worldwide with a significant economic impact: a recent estimate places it at the ten billion dollars/year. The control of HAIs requires a prompt and efficient identification of the etiological agent and a rapid communication with the clinician. The Microbiology Laboratory has a significant role in the prevention and control of these infections and is a key element of any Infection Control Program. The work of the Microbiology Laboratory covers microbial isolation and identification, determination of antimicrobial susceptibility patterns, epidemiological surveillance and outbreak detection, education, and report of quality assured results. In this paper we address the role and importance of the Microbiology Laboratory in the prevention and control of HAI and in Antibiotic Stewardship Programs and how it can be leveraged when combined with the use of information systems. Additionally, we critically review some challenges that the Microbiology Laboratory has to deal with, including the selection of analytic methods and the proper use of communication channels with other healthcare services.

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.

lytA-based identification methods can misidentify Streptococcus pneumoniae.

During surveillance studies we detected, among over 1500 presumptive pneumococci, 11 isolates displaying conflicting or novel results when characterized by widely accepted phenotypic (optochin susceptibility and bile solubility) and genotypic (lytA-BsaAI-RFLP and MLST) identification methods. We aimed to determine the genetic basis for the unexpected results given by lytA-BsaAI-RFLP and investigate the accuracy of the WHO recommended lytA real-time PCR assay to classify these 11 isolates. Three novel lytA-BsaAI-RFLP signatures were found (one in pneumococcus and two in S. mitis). In addition, one pneumococcus displayed the atypical lytA-BsaAI-RFLP signature characteristic of non-pneumococci and two S. pseudopneumoniae displayed the typical lytA-BsaAI-RFLP pattern characteristic of pneumococci. lytA real-time PCR misidentified these three isolates. In conclusion, identification of pneumococci by lytA real-time PCR, and other lytA-based methodologies, may lead to false results. This is of particular relevance in the increasingly frequent colonization studies relying solely on culture-independent methods.

The impact of private use of PCV7 in 2009 and 2010 on serotypes and antimicrobial resistance of Streptococcus pneumoniae carried by young children in Portugal: Comparison with data obtained since 1996 generating a 15-year study prior to PCV13 introduction.

In Portugal, the 7-valent pneumococcal conjugate vaccine (PCV7) was not introduced in the national immunization plan but was commercially available between 2001 and 2010. We studied serotype distribution and antibiotic susceptibility of Streptococcus pneumoniae carried by children in 2009 and 2010. Vaccination with PCV7 was extracted from children's immunization bulletins and information on recent antimicrobial consumption was obtained through a questionnaire. For comparison, we included data from previous studies conducted since 1996: 1996-1999, 2001-2003, 2006-2007. Pneumococci were isolated from nasopharyngeal samples of 1092 children up to six years old attending day-care in an urban area. Among these, 76% (819/1070) were vaccinated and 62% (677/1092) carried pneumococci. In 2009-2010, serotype replacement was extensive. Carriage of PCV7 serotypes was 4.9% and 5.8%, in 2009 and 2010, respectively, with the majority being of serotype 19F (carried by 4.3% and 4.6% of all participants, respectively). Colonization by serotype 19F was associated with vaccine status (7.7% (19/248) of non-vaccinees vs. 3.5% (29/818) of PCV7-vaccinees, p=0.010). Carriage of serotype 19A was high in 2009 and 2010 (8.6% of all participants) consistent with values already observed in 2007; carriage of serotype 6A was <1% (10/1092), indicating a major decline after 2007 (5.8% or 31/538, p<0.001). Non-vaccine serotypes increased and serotype 6C became the most frequently carried serotype in 2010 (11.2% (54/481)). High-level resistance to penicillin (MIC ≥2mg/L) showed a decreasing trend (p<0.001), whereas resistance to both penicillin and erythromycin increased (p<0.001) and was detected in 15-20% of all isolates in 2009-2010, most of which were non-vaccine serotypes. Antimicrobial use decreased over time (p<0.001). In conclusion, widespread private use of PCV7 has impacted on colonization leading to near elimination of all PCV7 serotypes except for serotype 19F. Antimicrobial consumption declined but it may be too soon to observe generalized changes in antimicrobial resistance rates.

Serotype Distribution, Antibiotic Resistance and Clonality of Streptococcus pneumoniae Isolated from Immunocompromised Patients in Tunisia.

BACKGROUND: Pneumococcal disease, a major cause of morbidity and mortality globally, has higher incidence among young children, the elderly and the immunocompromised of all ages. In Tunisia, pneumococcal conjugate vaccines (PCVs) are not included in the national immunization program. Also, few studies have described the epidemiology of S. pneumoniae in this country and, in particular, no molecular typing studies have been performed. The aim of this study was to evaluate serotype distribution, antimicrobial resistance and clonality of Streptococcus pneumoniae isolated from neutropenic patients in Tunisia. METHODS: Fifty-nine S. pneumoniae were isolated from infection (n = 31) and colonization (n = 28) sites of patients (children and adults) attending the National Centre of Bone Marrow Transplantation in Tunis between 2005-2011. All isolates were characterized by serotype, antimicrobial resistance pattern and multilocus sequence typing (MLST). RESULTS: The majority (66.1%) of the isolates belonged to five serotypes all included in PCVs: 6B, 9V, 14, 19F and 23F. The potential coverage of the 10-valent and 13-valent PCV was of 71.2% and 76.3% respectively. Resistance rates were very high and 69.5% of the isolates were multidrug resistant: non-susceptibility rates to penicillin, amoxicillin and cefotaxime were 66.1%, 40.7% and 27.1%, respectively; resistance rates to erythromycin, clindamycin, tetracycline, chloramphenicol and trimethoprim-sulfamethoxazole, were 69.5%, 61.0%, 37.3%, 22.0% and 67.8%, respectively. The most frequent serotypes had STs characteristic of multidrug resistant international clones known to be highly successful and important causes of pneumococcal infection: Spain 23F-ST81, France 9V/14-ST156, Spain 6B-ST90, 19F-ST320, and Portugal 19F-ST177. CONCLUSIONS: The majority of S. pneumoniae strains recovered from immunocompromised patients in Tunisia are representatives of multidrug resistant pandemic clones that express serotypes targeted by PCVs. To contain the burden of pneumococcal disease and improve treatment choices among Tunisian immunocompromised patients PCVs should be offered to all of them.

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.

Rapid identification of noncapsulated Streptococcus pneumoniae in nasopharyngeal samples allowing detection of co-colonization and reevaluation of prevalence.

Noncapsulated pneumococci are atypical Streptococcus pneumoniae that lack a capsule and therefore do not react with any available antisera. These isolates, which are often referred as nontypeable pneumococci (NTPn), are difficult to identify as their differentiation from closely related species such as Streptococcus pseudopneumoniae and other streptococcus of the mitis group is not always straightforward. We developed a low-cost and easy assay to detect and quantify NTPn in primary samples (which may contain multiple species) obtained from nasopharyngeal swabs. The strategy is based on a multiplex polymerase chain reaction targeting lytA, cpsA, aliB-like ORF2, and 16S rDNA genes, plus a restriction fragment length polymorphism assay to differentiate typical from atypical lytA. The application of the proposed methodology to over 500 nasopharyngeal samples found that the prevalence of NTPn in colonization was 3-fold higher than that estimated by routine methods (from 2.9% to 8.6% in the study collection). The international clone Norway(NT)ST344 was the major clone identified.

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.

Highly penicillin-resistant multidrug-resistant pneumococcus-like strains colonizing children in Oeiras, Portugal: genomic characteristics and implications for surveillance.

While performing surveillance studies in Oeiras, Portugal, designed to describe the impact of pneumococcal conjugate vaccine on colonization, we observed an increase from 0.7% in 2003 to 5% in 2006 in the prevalence of penicillin resistance (MIC of 2 to 6 mg/liter) among presumptively identified pneumococcal isolates. Although 15 of the 22 penicillin-resistant isolates obtained in 2006 were optochin resistant, they were bile soluble and thus considered to be bona fide pneumococci. This study aimed to clarify the nature of these isolates by using a combination of phenotypic and genotypic approaches that included routine strategies for pneumococcal identification, multilocus sequence analysis (MLSA), and comparative genomic hybridization (CGH). By MLSA, all isolates were classified as "streptococci of the mitis group" that, however, were distinct from typical Streptococcus pneumoniae or Streptococcus mitis. A single isolate was identified as Streptococcus pseudopneumoniae. CGH confirmed these findings and further indicated that a considerable part of the proposed pneumococcal core genome is conserved in these isolates, including several pneumococcal virulence genes (e.g., pavA, spxB, cbpE, and cbpD). These results suggest that among pneumococci and closely related streptococci, universal unique phenotypic and genetic properties that could aid species identification are virtually impossible to define. In pneumococcal colonization studies, when atypical strains are found, MLSA and CGH are informative tools that can be used to complement routine tests. In our study, after correct identification of the penicillin-resistant true pneumococci, we found that penicillin resistance levels among pneumococci remained stable from 2003 to 2006.

Identification, prevalence and population structure of non-typable Streptococcus pneumoniae in carriage samples isolated from preschoolers attending day-care centres.

The authors aimed to get insights into the population structure of non-(sero)typable pneumococci (NTPn), a specific group of natural atypical pneumococci whose identification is often difficult, and which has remained insufficiently studied. A total of 265 presumptive NTPn, isolated between 1997 and 2003 from the nasopharynx of children, were characterized. Strains were confirmed to be pneumococci on the basis of bile solubility, and PCR detection or Southern blotting hybridization of lytA and psaA, genes ubiquitous in this species. Multilocus sequence typing (MLST) was used to exclude two isolates that gave ambiguous results. Non-typability was confirmed by the Quellung reaction using Omniserum. A total of 213 isolates were considered to be true NTPn. The molecular analysis of the true NTPn by PFGE and MLST showed that this population was genetically diverse, although a dominant cluster, accounting for 66 % of the isolates, was identified. Antimicrobial resistance was observed in most genetic backgrounds, and multidrug resistance to penicillin, erythromycin, clindamycin, tetracycline and sulfamethoxazole-trimethoprim was associated with strains belonging to the dominant cluster. Comparison with PFGE fingerprints and sequence types of large collections of serotypable strains showed that the genetic backgrounds of all but two NTPn were different from those found in serotypable strains. In addition, we found that NTPn strains with similar genetic backgrounds to those identified in our study had been isolated from disease sources in other countries. These observations seem to indicate that NTPn have diverse genetic backgrounds and may have evolved as a distinct group of pneumococcal isolates.