Epidemiology of Clostridioides difficile Infection in Portugal: A Retrospective, Observational Study of Hospitalized Patients.

INTRODUCTION: Clostridioides difficile is the main cause of healthcare-associated diarrhea in Europe and North America. The aim of this study was to characterize the epidemiology and clinical burden of Clostridioides difficile infection among hospitalized patients in Portugal. MATERIAL AND METHODS: Retrospective study conducted in six public hospital centers in Portugal. All primary Clostridioides difficile infection episodes and related recurrences occurring in 2017, as well as episodes developing two to eight weeks after the last episode diagnosed in that year, were documented. The National Reference Laboratory (National Institute of Health Dr. Ricardo Jorge) provided national surveillance data on Clostridioides difficile infection. RESULTS: A total of 385 inpatients with at least one primary episode diagnosed in 2017 were included. Most patients were aged over 70 years-old (73.2%). The included patients developed 451 episodes during the observation period. Approximately 44% of primary episodes were community-associated. Most episodes (94.9%) occurred in patients with one or more risk factors, with recent antibiotic exposure being particularly common (86.0%). All-cause in-hospital mortality was 19.5%, being significantly higher in patients aged over 65 years-old versus those aged 18 to 64 years-old (22.4% vs 7.8%, respectively). Over 50 different ribotypes were observed among 206 Clostridioides difficile strains received by the National Reference Laboratory. CONCLUSION: In Portugal, hospitalized patients with Clostridioides difficile infection are mostly older patients presenting risk factors for the development of this infection, particularly recent antibiotic exposure. Mortality is disproportionately high among the older population. Community-associated Clostridioides difficile infection is common among inpatients with this infection.

Introdução: Clostridioides difficile é a principal causa de diarreia nosocomial na Europa e América do Norte. Este estudo teve como objetivo caracterizar a epidemiologia e o impacto clínico da infeção por Clostridioides difficile em doentes hospitalizados em Portugal. Material e Métodos: Estudo retrospetivo conduzido em seis centros hospitalares públicos de Portugal. Foram documentados todos os episódios primários de infeção por Clostridioides difficile ocorridos em 2017 e consequentes recorrências, bem como os episódios que ocorreram entre duas a oito semanas após o último episódio diagnosticado neste ano. Os dados de vigilância nacional de infeção por Clostridioides difficile foram fornecidos pelo laboratório nacional de referência (Instituto Nacional de Saúde Doutor Ricardo Jorge). Resultados: Foram incluídos 385 doentes hospitalizados com pelo menos um episódio primário diagnosticado em 2017. A maioria dos doentes tinha idade igual ou superior a 70 anos (73,2%). Os doentes incluídos tiveram 451 episódios durante o período de observação. Aproximadamente 44% dos episódios primários eram episódios de infeção por Clostridioides difficile adquirida na comunidade. A maioria dos episódios (91,8%) ocorreu em doentes com um ou mais fatores de risco, sendo a exposição recente a antibióticos particularmente comum (86,0%). A mortalidade hospitalar por todas as causas foi de 19,5%, sendo significativamente superior em doentes com idade igual ou superior a 65 anos comparativamente a doentes com idade entre 18 e 64 anos (22,4% versus 7,8%, respetivamente). Mais de 50 ribotipos diferentes foram detetados entre as 206 estirpes de Clostridioides difficile recebidas pelo laboratório nacional de referência. Conclusão: Em Portugal, doentes hospitalizados com infeção por Clostridioides difficile são, na sua maioria, doentes idosos com fatores de risco para o seu desenvolvimento, particularmente exposição recente a antibióticos. A mortalidade é desproporcionalmente elevada na população idosa. Episódios associados à comunidade são comuns em doentes hospitalizados com esta infeção.

Real-World Characterization of the Portuguese Population Living with HIV who Initiated Raltegravir Based-Regimens: The REALITY Study.

INTRODUCTION: Although raltegravir has been available since 2007, data are lacking on the Portuguese population living with HIV who initiated this antiretroviral therapy. Hence, this study aimed to characterize the patients who initiated raltegravir-based regimens between January 2015 and December 2017, on sociodemographics, clinical features, and treatment satisfaction. MATERIAL AND METHODS: Observational, retrospective, multicentre study conducted at 11 reference sites. Sociodemographic and clinical data were collected retrospectively from hospital medical records. For participants continuing raltegravir at study inclusion, the HIV Treatment Satisfaction Questionnaire was administered to assess satisfaction with raltegravir-based therapy. Descriptive statistics were performed. Treatment-naïve and treatment-experienced subgroups were compared for demographic and clinical variables. RESULTS: A total of 302 patients were included; mostly men (69.5%) with a mean age of 49 years old. Approximately half of the patients had at least one non-AIDS-related comorbidity at baseline (53.3%), such as hypercholesterolemia, arterial hypertension, diabetes mellitus, and depression. Moreover, 52.3% were treatment-experienced patients with up to two treatments prior to raltegravir. Across the study time points, there was a reduction in the viral load and improvement in CD4 counts in both the treatment-naïve and treatment-experienced subgroups. Continuing users of raltegravir reported high treatment satisfaction (55.4 ± 7.2 points). CONCLUSION: Raltegravir-based regimens seem like a valid therapeutic option in heterogeneous populations of HIV-infected patients, in patients with previous ART experience and as part of first-line therapeutic options alongside with the latest generation of drugs from its class.

Introdução: Apesar de o raltegravir estar disponível desde 2007, os dados na população portuguesa com VIH que iniciou esta terapêutica antirretroviral são escassos. Deste modo, este estudo teve por objetivo caracterizar os doentes que iniciaram um regime terapêutico baseado em raltegravir entre janeiro de 2015 e dezembro de 2017, relativamente a dados sociodemográficos, características clínicas e satisfação com o tratamento. Material e Métodos: Estudo observacional, retrospetivo, multicêntrico conduzido em 11 centros de referência. Os dados sociodemográficos e clínicos foram recolhidos retrospetivamente nos processos clínicos. Os participantes que continuaram o regime com raltegravir após a inclusão no estudo preencheram o HIV Treatment Satisfaction Questionnaire para avaliar a satisfação com a terapêutica. Foram efetuadas análises de estatística descritiva e comparações para as variáveis sociodemográficas e clínicas nos subgrupos de doentes naïve de tratamento e de doentes com experiência terapêutica. Resultados: Foram incluídos 302 doentes, maioritariamente do sexo masculino (69,5%) com idade média de 49 anos. Aproximadamente metade dos doentes tinha pelo menos uma comorbilidade não relacionada com SIDA no início do estudo (53,3%), tais como hipercolesterolemia, hipertensão arterial, diabetes mellitus ou depressão. Adicionalmente, 52,3% eram doentes com experiência terapêutica com até dois tratamentos anteriores ao raltegravir. Ao longo do estudo verificou-se uma redução na carga viral e uma melhoria nas contagens de CD4 em ambos os subgrupos de doentes (doentes naïve de tratamento e doentes com experiência terapêutica). Os doentes com uso continuado de raltegravir reportaram uma elevada satisfação com o tratamento (55,4 ± 7,2 pontos). Conclusão: Os regimes terapêuticos baseados em raltegravir parecem ser uma opção terapêutica válida em populações heterogéneas de doentes infetados com VIH, em doentes com experiência em ART e como tratamento de primeira linha, em paralelo com outras terapêuticas de última geração.

Presence of Chromosomal crpP-like Genes Is Not Always Associated with Ciprofloxacin Resistance in Pseudomonas aeruginosa Clinical Isolates Recovered in ICU Patients from Portugal and Spain.

CrpP enzymes have been recently described as a novel ciprofloxacin-resistance mechanism. We investigated by whole genome sequencing the presence of crpP-genes and other mechanisms involved in quinolone resistance in MDR/XDR-Pseudomonas aeruginosa isolates (n = 55) with both ceftolozane-tazobactam susceptible or resistant profiles recovered from intensive care unit patients during the STEP (Portugal) and SUPERIOR (Spain) surveillance studies. Ciprofloxacin resistance was associated with mutations in the gyrA and parC genes. Additionally, plasmid-mediated genes (qnrS2 and aac(6')-Ib-cr) were eventually detected. Ten chromosomal crpP-like genes contained in related pathogenicity genomic islands and 6 different CrpP (CrpP1-CrpP6) proteins were found in 65% (36/55) of the isolates. Dissemination of CrpP variants was observed among non-related clones of both countries, including the CC175 (Spain) high-risk clone and CC348 (Portugal) clone. Interestingly, 5 of 6 variants (CrpP1-CrpP5) carried missense mutations in an amino acid position (Gly7) previously defined as essential conferring ciprofloxacin resistance, and decreased ciprofloxacin susceptibility was only associated with the novel CrpP6 protein. In our collection, ciprofloxacin resistance was mainly due to chromosomal mutations in the gyrA and parC genes. However, crpP genes carrying mutations essential for protein function (G7, I26) and associated with a restored ciprofloxacin susceptibility were predominant. Despite the presence of crpP genes is not always associated with ciprofloxacin resistance, the risk of emergence of novel CrpP variants with a higher ability to affect quinolones is increasing. Furthermore, the spread of crpP genes in highly mobilizable genomic islands among related and non-related P. aeruginosa clones alert the dispersion of MDR pathogens in hospital settings.

Confronting Ceftolozane-Tazobactam Susceptibility in Multidrug-Resistant Enterobacterales Isolates and Whole-Genome Sequencing Results (STEP Study).

Ceftolozane-tazobactam (C/T) is frequently used for infections caused by multidrug-resistant (MDR)-Enterobacterales isolates. Whole-genome sequencing (WGS, Illumina-Hiseq 4000/NovaSeq 6000, OGC, UK) was used to study the population structure, the resistome and the virulome of C/T-susceptible and -resistant MDR Escherichia spp. (n=30) and Klebsiella spp. (n=78) isolates, recovered from lower respiratory, intra-abdominal and urinary tract infections of ICU patients from 11 Portuguese Hospitals (STEP study, 2017-2018). Minimum inhibitory concentrations (MICs) were determined (ISO-broth microdilution, breakpoints EUCAST-2020). In Escherichia spp., a weak concordance between the phenotypic and the WGS method (P=0.051) was observed in the carbapenemase detection (3/30) [blaVIM-2 (2/3), blaKPC-3 (1/3)]; VIM-2-Escherichia coli isolates were C/T-susceptible and only the KPC-3-Escherichia marmotae producer showed C/T-resistance. Overall, CTX-M-15-E. coli-ST131-O25:H4-H30-Rx (11/30) was the most frequent subclone, followed by CTX-M-27-E. coli-ST131-O25:H4-H30 (4/4). Moreover, a wide resistome and virulome were detected in all E. coli isolates. Among Klebsiella spp. isolates [K. pneumoniae (67/78), K. aerogenes (7/78), K. oxytoca (2/78), K. variicola (2/78)], concordance (P<0.001) was observed between the phenotypic and the genomic carbapenemase detection (21/78) [blaKPC-3 (14/21), blaOXA-48 (3/21), blaOXA-181 (3/21)]. A high correlation between C/T-resistance and carbapenemase detection was established (P<0.05). Overall, a high clonal diversity was observed, mainly in KPC-3-producing K. pneumoniae isolates. An extensive resistome was detected in Klebsiella spp. isolates, whereas virulence determinants were mostly identified in carbapenemase producers (P<0.001). WGS is a powerful tool for typing characterization and microbiological study of MDR-Enterobacterales pathogens. Furthermore, carbapenemase genes are associated with C/T-resistance in Klebsiella spp., but other mechanisms might also be involved.

Distinct epidemiology and resistance mechanisms affecting ceftolozane/tazobactam in Pseudomonas aeruginosa isolates recovered from ICU patients in Spain and Portugal depicted by WGS.

OBJECTIVES: To analyse the epidemiology, the resistome and the virulome of ceftolozane/tazobactam-susceptible or -resistant Pseudomonas aeruginosa clinical isolates recovered from surveillance studies in Portugal (STEP, 2017-18) and Spain (SUPERIOR, 2016-17). METHODS: P. aeruginosa isolates were recovered from intra-abdominal, urinary tract and lower respiratory tract infections in ICU patients admitted to 11 Portuguese and 8 Spanish hospitals. MICs were determined (ISO-standard broth microdilution, EUCAST 2020 breakpoints). A subset of 28 ceftolozane/tazobactam-resistant P. aeruginosa isolates were analysed and compared with 28 ceftolozane/tazobactam-susceptible P. aeruginosa strains by WGS. RESULTS: Clonal complex (CC) 235 (27%) and CC175 (18%) were the most frequent, followed by CC244 (13%), CC348 (9%), CC253 (5%) and CC309 (5%). Inter-hospital clonal dissemination was observed, limited to a geographical region (CC235, CC244, CC348 and CC253 in Portugal and CC175 and CC309 in Spain). Carbapenemases were detected in 25 isolates (45%): GES-13 (13/25); VIM type (10/25) [VIM-2 (4/10), VIM-20 (3/10), VIM-1 (2/10) and VIM-36 (1/10)]; and KPC-3 (2/25). GES-13-CC235 (13/15) and VIM type-CC175 (5/10) associations were observed. Interestingly, KPC-3 and VIM-36 producers showed ceftolozane/tazobactam-susceptible phenotypes. However, ceftolozane/tazobactam resistance was significantly associated with GES-13 and VIM-type carbapenemase production. Six non-carbapenemase producers also displayed ceftolozane/tazobactam resistance, three of them showing known ceftolozane/tazobactam resistance-associated mutations in the PBP3 gene, ftsI (R504C and F533L). Overall, an extensive virulome was identified in all P. aeruginosa isolates, particularly in carbapenemase-producing strains. CONCLUSIONS: GES-13-CC235 and VIM type-CC175 were the most frequent MDR/XDR P. aeruginosa clones causing infections in Portuguese and Spanish ICU patients, respectively. Ceftolozane/tazobactam resistance was mainly due to carbapenemase production, although mutations in PBP-encoding genes may additionally be involved.

In vitro activity of ceftolozane-tazobactam against Enterobacterales and Pseudomonas aeruginosa causing urinary, intra-abdominal and lower respiratory tract infections in intensive care units in Portugal: The STEP multicenter study.

The STEP surveillance study was designed to increase knowledge about distribution of multidrug-resistant (MDR) Enterobacterales and Pseudomonas aeruginosa in Portugal, focusing on the intensive care unit (ICU). Antimicrobial susceptibility of common agents was also evaluated and compared with that of one of the latest therapeutic introductions, ceftolozane-tazobactam (C/T). Clinical isolates of Enterobacterales (n=426) and P. aeruginosa (n=396) from patients admitted in Portuguese ICUs were included. Activity of C/T and comparators was investigated using standard broth microdilution. Isolates were recovered from urinary tract (UTI, 36.9%), intra-abdominal (IAI, 24.2%) and lower respiratory tract (LRTI, 38.9%) infections. In P. aeruginosa, overall distribution of MDR/extremely-drug resistant (XDR)/pan-drug resistant (PDR) isolates accounted for 21.2%, 23.2% and 0.8%, respectively. C/T was the most potent agent tested against P. aeruginosa and MDR/XDR/PDR phenotypes. In Escherichia coli, extended-spectrum beta-lactamases (ESBL) and carbapenemase (CP) phenotypes accounted for 16.6% and 1.7%, respectively, whereas in Klebsiella spp., ESBL and CP-phenotypes represented 28.5% and 17.9%, respectively. Overall, susceptibility of C/T against Enterobacterales was 86.9%. C/T was the least affected agent in E. coli (99.4% susceptibility), whereas its activity was moderate in Klebsiella spp. (71.5%) and Enterobacter spp. (70.4%), due in part to a high rate of ESBL and CP-phenotypes. In Enterobacterales, blaKPC was the most prevalent CP gene (63.0%), followed by blaOXA-48 (33.3%) and blaVIM (3.7%). These microbiological results reinforce C/T as a therapeutic option in ICU patients with UTI, IAI or LRTI due to P. aeruginosa or Enterobacterales isolates, but not for CP producers.

Differential Kinetics in Alteration and Recovery of Cognitive Processes from a Chronic Sleep Restriction in Young Healthy Men.

Chronic sleep restriction (CSR) induces neurobehavioral deficits in young and healthy people with a morning failure of sustained attention process. Testing both the kinetic of failure and recovery of different cognitive processes (i.e., attention, executive) under CSR and their potential links with subject's capacities (stay awake, baseline performance, age) and with some biological markers of stress and anabolism would be useful in order to understand the role of sleep debt on human behavior. Twelve healthy subjects spent 14 days in laboratory with 2 baseline days (B1 and B2, 8 h TIB) followed by 7 days of sleep restriction (SR1-SR7, 4 h TIB), 3 sleep recovery days (R1-R3, 8 h TIB) and two more ones 8 days later (R12-R13). Subjective sleepiness (KSS), maintenance of wakefulness latencies (MWT) were evaluated four times a day (10:00, 12:00 a.m. and 2:00, 4:00 p.m.) and cognitive tests were realized at morning (8:30 a.m.) and evening (6:30 p.m.) sessions during B2, SR1, SR4, SR7, R2, R3 and R13. Saliva (B2, SR7, R2, R13) and blood (B1, SR6, R1, R12) samples were collected in the morning. Cognitive processes were differently impaired and recovered with a more rapid kinetic for sustained attention process. Besides, a significant time of day effect was only evidenced for sustained attention failures that seemed to be related to subject's age and their morning capacity to stay awake. Executive processes were equally disturbed/recovered during the day and this failure/recovery process seemed to be mainly related to baseline subject's performance and to their capacity to stay awake. Morning concentrations of testosterone, cortisol and α-amylase were significantly decreased at SR6-SR7, but were either and respectively early (R1), tardily (after R2) and not at all (R13) recovered. All these results suggest a differential deleterious and restorative effect of CSR on cognition through biological changes of the stress pathway and subject's capacity (ClinicalTrials-NCT01989741).

Transcriptional and metabolic effects of glucose on Streptococcus pneumoniae sugar metabolism.

Streptococcus pneumoniae is a strictly fermentative human pathogen that relies on carbohydrate metabolism to generate energy for growth. The nasopharynx colonized by the bacterium is poor in free sugars, but mucosa lining glycans can provide a source of sugar. In blood and inflamed tissues glucose is the prevailing sugar. As a result during progression from colonization to disease S. pneumoniae has to cope with a pronounced shift in carbohydrate nature and availability. Thus, we set out to assess the pneumococcal response to sugars found in glycans and the influence of glucose (Glc) on this response at the transcriptional, physiological, and metabolic levels. Galactose (Gal), N-acetylglucosamine (GlcNAc), and mannose (Man) affected the expression of 8 to 14% of the genes covering cellular functions including central carbon metabolism and virulence. The pattern of end-products as monitored by in vivo (13)C-NMR is in good agreement with the fermentation profiles during growth, while the pools of phosphorylated metabolites are consistent with the type of fermentation observed (homolactic vs. mixed) and regulation at the metabolic level. Furthermore, the accumulation of α-Gal6P and Man6P indicate metabolic bottlenecks in the metabolism of Gal and Man, respectively. Glc added to cells actively metabolizing other sugar(s) was readily consumed and elicited a metabolic shift toward a homolactic profile. The transcriptional response to Glc was large (over 5% of the genome). In central carbon metabolism (most represented category), Glc exerted mostly negative regulation. The smallest response to Glc was observed on a sugar mix, suggesting that exposure to varied sugars improves the fitness of S. pneumoniae. The expression of virulence factors was negatively controlled by Glc in a sugar-dependent manner. Overall, our results shed new light on the link between carbohydrate metabolism, adaptation to host niches and virulence.

Host glycan sugar-specific pathways in Streptococcus pneumoniae: galactose as a key sugar in colonisation and infection [corrected].

The human pathogen Streptococcus pneumoniae is a strictly fermentative organism that relies on glycolytic metabolism to obtain energy. In the human nasopharynx S. pneumoniae encounters glycoconjugates composed of a variety of monosaccharides, which can potentially be used as nutrients once depolymerized by glycosidases. Therefore, it is reasonable to hypothesise that the pneumococcus would rely on these glycan-derived sugars to grow. Here, we identified the sugar-specific catabolic pathways used by S. pneumoniae during growth on mucin. Transcriptome analysis of cells grown on mucin showed specific upregulation of genes likely to be involved in deglycosylation, transport and catabolism of galactose, mannose and N acetylglucosamine. In contrast to growth on mannose and N-acetylglucosamine, S. pneumoniae grown on galactose re-route their metabolic pathway from homolactic fermentation to a truly mixed acid fermentation regime. By measuring intracellular metabolites, enzymatic activities and mutant analysis, we provide an accurate map of the biochemical pathways for galactose, mannose and N-acetylglucosamine catabolism in S. pneumoniae. Intranasal mouse infection models of pneumococcal colonisation and disease showed that only mutants in galactose catabolic genes were attenuated. Our data pinpoint galactose as a key nutrient for growth in the respiratory tract and highlights the importance of central carbon metabolism for pneumococcal pathogenesis.

BGFit: management and automated fitting of biological growth curves.

BACKGROUND: Existing tools to model cell growth curves do not offer a flexible integrative approach to manage large datasets and automatically estimate parameters. Due to the increase of experimental time-series from microbiology and oncology, the need for a software that allows researchers to easily organize experimental data and simultaneously extract relevant parameters in an efficient way is crucial. RESULTS: BGFit provides a web-based unified platform, where a rich set of dynamic models can be fitted to experimental time-series data, further allowing to efficiently manage the results in a structured and hierarchical way. The data managing system allows to organize projects, experiments and measurements data and also to define teams with different editing and viewing permission. Several dynamic and algebraic models are already implemented, such as polynomial regression, Gompertz, Baranyi, Logistic and Live Cell Fraction models and the user can add easily new models thus expanding current ones. CONCLUSIONS: BGFit allows users to easily manage their data and models in an integrated way, even if they are not familiar with databases or existing computational tools for parameter estimation. BGFit is designed with a flexible architecture that focus on extensibility and leverages free software with existing tools and methods, allowing to compare and evaluate different data modeling techniques. The application is described in the context of bacterial and tumor cells growth data fitting, but it is also applicable to any type of two-dimensional data, e.g. physical chemistry and macroeconomic time series, being fully scalable to high number of projects, data and model complexity.

Fluorometric determination of ethidium bromide efflux kinetics in Escherichia coli.

BACKGROUND: Efflux pump activity has been associated with multidrug resistance phenotypes in bacteria, compromising the effectiveness of antimicrobial therapy. The development of methods for the early detection and quantification of drug transport across the bacterial cell wall is a tool essential to understand and overcome this type of drug resistance mechanism. This approach was developed to study the transport of the efflux pump substrate ethidium bromide (EtBr) across the cell envelope of Escherichia coli K-12 and derivatives, differing in the expression of their efflux systems. RESULTS: EtBr transport across the cell envelope of E. coli K-12 and derivatives was analysed by a semi-automated fluorometric method. Accumulation and efflux of EtBr was studied under limiting energy supply (absence of glucose and low temperature) and in the presence and absence of the efflux pump inhibitor, chlorpromazine. The bulk fluorescence variations were also observed by single-cell flow cytometry analysis, revealing that once inside the cells, leakage of EtBr does not occur and that efflux is mediated by active transport. The importance of AcrAB-TolC, the main efflux system of E. coli, in the extrusion of EtBr was evidenced by comparing strains with different levels of AcrAB expression. An experimental model was developed to describe the transport kinetics in the three strains. The model integrates passive entry (influx) and active efflux of EtBr, and discriminates different degrees of efflux between the studied strains that vary in the activity of their efflux systems, as evident from the calculated efflux rates: = 0.0173 +/- 0.0057 min-1; = 0.0106 +/- 0.0033 min-1; and = 0.0230 +/- 0.0075 min-1. CONCLUSION: The combined use of a semi-automated fluorometric method and an experimental model allowed quantifying EtBr transport in E. coli strains that differ in their overall efflux activity. This methodology can be used for the early detection of differences in the drug efflux capacity in bacteria accounting for antibiotic resistance, as well as for expedite screening of new drug efflux inhibitors libraries and transport studies across the bacterial cell wall.

Demonstration of intrinsic efflux activity of Escherichia coli K-12 AG100 by an automated ethidium bromide method.

Demonstration of efflux of ethidium bromide (EtBr) has been made for over 30 bacterial species, usually by showing enhanced efflux in multidrug-resistant strains that was then abolished by inactivating efflux pumps. Here we present a relatively simple automated method that employs EtBr as an efflux pump substrate for the demonstration of intrinsic efflux activity in Escherichia coli K-12 AG100. The method uses the Rotor-Gene 3000 instrument for real-time fluorometric measurement of EtBr accumulation under conditions that limit energy (absence of glucose, low temperature) and of EtBr extrusion under optimum conditions. The method can be used for screening compound libraries for efflux inhibiting capacity.