Percorrer por autor "Pinto, Margarida"
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- Genome-wide analysis and longitudinal study of Klebsiella pneumoniae in Portugal : tracing the evolution and spread of carbapenem resistancePublication . Elias, Rita; Phelan, Jody E.; Lito, Luís; Caneiras, Cátia; Marques, Cátia; Pinto, Margarida; Cavaco-Silva, Patrícia; Ferreira, Helena; Pomba, Constança; Silva, Gabriela J. da; Saavedra, Maria José; Coelho, Rosário; Lourinho, Rita; Gonçalves, Luísa; Hinthong, Woranich; Rosa, Maria João; Melo-Cristino, José; Campino, Susana; Portugal, Isabel; Duarte, Aida; Perdigão, JoãoBackground: Carbapenem-resistant Klebsiella pneumoniae (CRKP) has high incidence in Portugal, causing severe and often fatal infections. Objectives: Characterize the evolutionary history and epidemiology of CRKP in Portugal over a 40-year period. Methods: WGS was performed using the Illumina platform. In silico multilocus sequence typing, surface antigen characterization, and resistance gene detection were subsequently carried out. Core and pan-genome analyses were conducted using Roary. Genomic clusters (GCs) were identified based on a 21-SNP threshold. To estimate the divergence times of the most prevalent sequence types (ST) in the dataset, Bayesian evolutionary analysis was performed using BEAST. Results: Nineteen GCs harboring carbapenemases were identified. The blaKPC-3 gene was the most prevalent carbapenemase, linked to strains circulating in both hospital and community settings, with dissemination patterns at regional, interregional, and international levels. ST15 was the most established sequence type in Portugal, with nine distinct GCs identified in both clinical and environmental samples. Towards the end of 2010s, ST147 and ST13 were responsible for significant outbreaks associated with blaKPC-3. Conclusions: This study underscores the value of genomic-based surveillance in understanding the evolution of high-risk clones coupled with the spread of AMR determinants. The data obtained highlights a shift in ST predominance across the country from an ST15-dominated period and strongly associated with ESBL dissemination, to the emergence of ST147 and ST13 CRKP clones, the latter associated with international transmission. This work further stresses the importance of cross-border surveillance efforts to monitor the emergence and dissemination of CRKP strains and inform risk assessment and prevention.
- Genomic epidemiological analysis of Klebsiella pneumoniae from Portuguese hospitals reveals insights into circulating antimicrobial resistancePublication . Spadar, Anton; Phelan, Jody; Elias, Rita; Modesto, Ana; Caneiras, Cátia; Marques, Cátia; Lito, Luís; Pinto, Margarida; Cavaco-Silva, Patrícia; Ferreira, Helena; Pomba, Constança; Silva, Gabriela J. Da; Saavedra, Maria José; Melo-Cristino, José; Duarte, Aida; Campino, Susana; Perdigão, João; Clark, Taane G.Klebsiella pneumoniae (Kp) bacteria are an increasing threat to public health and represent one of the most concerning pathogens involved in life-threatening infections and antimicrobial resistance (AMR). To understand the epidemiology of AMR of Kp in Portugal, we analysed whole genome sequencing, susceptibility testing and other meta data on 509 isolates collected nationwide from 16 hospitals and environmental settings between years 1980 and 2019. Predominant sequence types (STs) included ST15 (n = 161, 32%), ST147 (n = 36, 7%), ST14 (n = 26, 5%) or ST13 (n = 26, 5%), while 31% of isolates belonged to STs with fewer than 10 isolates. AMR testing revealed widespread resistance to aminoglycosides, fluoroquinolones, cephalosporins and carbapenems. The most common carbapenemase gene was blaKPC-3. Whilst the distribution of AMR linked plasmids appears uncorrelated with ST, their frequency has changed over time. Before year 2010, the dominant plasmid group was associated with the extended spectrum beta-lactamase gene blaCTX-M-15, but this group appears to have been displaced by another carrying the blaKPC-3 gene. Co-carriage of blaCTX-M and blaKPC-3 was uncommon. Our results from the largest genomics study of Kp in Portugal highlight the active transmission of strains with AMR genes and provide a baseline set of variants for future resistance monitoring and epidemiological studies.
- Klebsiella pneumoniae and colistin susceptibility testing : performance evaluation for broth microdilution, agar dilution and minimum inhibitory concentration test strips and impact of the “skipped well” phenomenonPublication . Elias, Rita; Melo-Cristino, José; Lito, Luís; Pinto, Margarida; Gonçalves, Luísa; Campino, Susana; Clark, Taane G.; Duarte, Aida; Perdigão, JoãoThe emergence of multidrug resistant Gram-negative pathogens, particularly carbapenemase producers, has forced clinicians to use last line antibiotics, such as colistin. Since colistin susceptibility testing presents several challenges, this study aimed at evaluating the performance of two alternative susceptibility methods for Klebsiella pneumoniae, namely, agar dilution (AD) and MIC test strips (MTS). These approaches were compared with the reference method, broth microdilution (BMD), and provide a quantitative description for the “skipped well” (SW) phenomenon. Colistin susceptibility was evaluated by BMD and AD in parallel and triplicate, using 141 K. pneumoniae clinical isolates while MTS performance was evaluated only for a subset (n = 121). Minimum inhibitory concentration analysis revealed that a substantial part (n = 26/141; 18.4%) of the initial isolates was deemed undetermined by BMD due to the following: discordance between replicates (1.4%); presence of multiple SWs (7.8%); and the combination of both events (9.2%). Both AD and MTS revealed a high number of false-susceptible strains (“very major errors”), 37.5% and 68.8%, respectively. However, AD agreement indices were reasonably high (EA = 71.3% and CA = 94.8%). For MTS these indices were lower, in particular EA (EA = 41.7% and CA = 89.6), but the approach enabled the detection of distinct sub-populations for four isolates. In conclusion, this study provides the most comprehensive study on the performance of AD and MTS for colistin susceptibility testing in K. pneumoniae, highlighting its limitations, and stressing the importance of sample size and composition. Further, this study highlights the impact of the SW phenomenon associated with the BMD method for K. pneumoniae.
- A phylogenomic approach for the analysis of colistin resistance-associated genes in Klebsiella pneumoniae, its mutational diversity and implications for phenotypic resistancePublication . Elias, Rita; Spadar, Anton; Phelan, Jody; Melo-Cristino, José; Lito, Luís; Pinto, Margarida; Gonçalves, Luísa; Campino, Susana; Clark, Taane G.; Duarte, Aida; Perdigão, JoãoThe emergence of carbapenemase-producing Klebsiella pneumoniae strains has triggered the use of old antibiotics such as colistin. This is driving the emergence of colistin resistance in multidrug-resistant strains that underlie life-threatening infections. This study analyses the mutational diversity of 22 genes associated with colistin resistance in 140 K. pneumoniae clinical isolates integrated in a high-resolution phylogenetic scenario. Colistin susceptibility was accessed by broth microdilution. A total of 98 isolates were susceptible and 16 were resistant, 10 of which were carbapenemase producers. Across the 22 genes examined, 171 non-synonymous mutations and 9 mutations associated with promoter regions were found. Eighty-five isolates had a truncation and/or deletion in at least one of the 22 genes. However, only seven mutations, the complete deletion of mgrB or insertion sequence (IS)-mediated disruption, were exclusively observed in resistant isolates. Four of these (mgrB Ile13fs, pmrB Gly207Asp, phoQ His339Asp and ramA Ile28Met) comprised novel mutations that are potentially involved in colistin resistance. One strain bore a ISEcp1-blaCTX-M-15::mgrB disruption, underlying co-resistance to third-generation cephalosporins and colistin. Moreover, the high-resolution phylogenetic context shows that most of the mutational diversity spans multiple phylogenetic clades, and most of the mutations previously associated with colistin resistance are clade-associated and present in susceptible isolates, showing no correlation with colistin resistance. In conclusion, the present study provides relevant data on the genetic background of genes involved with colistin resistance deeply rooted across monophyletic groups and provides a better understanding of the genes and mutations involved in colistin resistance.
- Rir para curar!: operação nariz vermelhoPublication . Moreira, Sandrina Berthault; Reis, Tatiana; Moreira, Rodrigo; Paiva, Ana; Pinto, Margarida; Cunha, Samuel