Percorrer por autor "Melo-Cristino, José"
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- First description of Ceftazidime/Avibactam resistance in a ST13 KPC-70-producing Klebsiella pneumoniae strain from PortugalPublication . Mendes, Gabriel; Ramalho, João F.; Bruschy-Fonseca, Ana; Lito, Luís; Duarte, Aida; Melo-Cristino, José; Caneiras, CátiaThe combination of ceftazidime/avibactam (CZA) is a novel β-lactam/β-lactamase inhibitor with activity against Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacterales. Emerging cases caused by CZA-resistant strains that produce variants of KPC genes have already been reported worldwide. However, to the best of our knowledge, no CZA-resistant strains were reported in Portugal. In September 2019, a K. pneumoniae CZA-resistant strain was collected from ascitic fluid at a surgery ward of a tertiary University Hospital Center in Lisboa, Portugal. The strain was resistant to ceftazidime/avibactam, as well as to ceftazidime, cefoxitin, gentamicin, amoxicillin/clavulanic acid, and ertapenem, being susceptible to imipenem and tigecycline. A hypermucoviscosity phenotype was confirmed by string test. Whole-genome sequencing (WGS) analysis revealed the presence of an ST13 KPC70-producing K. pneumoniae, a KPC-3 variant, differing in two amino-acid substitutions (D179Y and T263A). The D179Y mutation in the KPC Ω-loop region is the most common amino-acid substitution in KPC-2 and KPC-3, further leading to CZA resistance. The second mutation causes a KPC-70 variant in which threonine replaces alanine (T263A). The CZA-resistant strain showed the capsular locus KL3 and antigen locus O1v2. Other important virulence factors were identified: fimbrial adhesins type 1 and type 3, as well as the cluster of iron uptake systems aerobactin, enterobactin, salmochelin, and yersiniabactin included in integrative conjugative element 10 (ICEKp10) with the genotoxin colibactin cluster. Herein, we report the molecular characterization of the first hypervirulent CZA-resistant ST13 KPC-70-producing K. pneumoniae strain in Portugal. The emergence of CZA-resistant strains might pose a serious threat to public health and suggests an urgent need for enhanced clinical awareness and epidemiologic surveillance.
- 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.
- In vitro design of a novel lytic bacteriophage cocktail with therapeutic potential against organisms causing diabetic foot infectionsPublication . Mendes, João J.; Leandro, Clara; Mottola, Carla; Barbosa, Raquel; Silva, Filipa A.; Oliveira, Manuela; Vilela, Cristina L.; Melo-Cristino, José; Górski, Andrzej; Pimentel, Madalena; São-José, Carlos; Cavaco-Silva, Patrícia; Garcia, MiguelIn patients with diabetes mellitus, foot infections pose a significant risk. These are complex infections commonly caused by Staphylococcus aureus, Pseudomonas aeruginosa and Acinetobacter baumannii, all of which are potentially susceptible to bacteriophages. Here, we characterized five bacteriophages that we had determined previously to have antimicrobial and wound-healing potential in chronic S. aureus, P. aeruginosa and A. baumannii infections. Morphological and genetic features indicated that the bacteriophages were lytic members of the family Myoviridae or Podoviridae and did not harbour any known bacterial virulence genes. Combinations of the bacteriophages had broad host ranges for the different target bacterial species. The activity of the bacteriophages against planktonic cells revealed effective, early killing at 4 h, followed by bacterial regrowth to pre-treatment levels by 24 h. Using metabolic activity as a measure of cell viability within established biofilms, we found significant cell impairment following bacteriophage exposure. Repeated treatment every 4 h caused a further decrease in cell activity. The greatest effects on both planktonic and biofilm cells occurred at a bacteriophage : bacterium input multiplicity of 10. These studies on both planktonic cells and established biofilms allowed us to better evaluate the effects of a high input multiplicity and a multiple-dose treatment protocol, and the findings support further clinical development of bacteriophage therapy.
- 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.
- Whole-genome sequencing enables molecular characterization of non-clonal group 258 high-risk clones (ST13, ST17, ST147 and ST307) among carbapenem-resistant Klebsiella pneumoniae from a tertiary university hospital centre in PortugalPublication . Mendes, Gabriel; Ramalho, João F.; Bruschy-Fonseca, Ana; Lito, Luís; Duarte, Aida; Melo-Cristino, José; Caneiras, CátiaThe carbapenem-resistant Enterobacterales (CRE) strains have been identified by the World Health Organization as critical priority pathogens in research and development of diagnostics, treatments, and vaccines. However, recent molecular information about carbapenem-resistant K. pneumoniae (CRK) epidemiology in Portugal is still scarce. Thus, this study aimed to provide the molecular epidemiology, resistome, and virulome of CRK clinical strains recovered from a tertiary care hospital centre (2019–2021) using polymerase chain reaction (PCR) and the advanced molecular technique whole-genome sequencing (WGS). PCR amplification of carbapenemase genes was performed in 437 carbapenem-resistant K. pneumoniae strains. The most frequent carbapenemases were: KPC-3 (42%), followed by OXA-181 (20%), GES-5 (0.2%), and NDM-1 (0.2%). Additionally, 10 strains (2%) coproduced KPC-3 and OXA-181, and 1 strain coproduced KPC-3 and OXA-48 (0.2%). The genomic population structure of 68 strains characterized by WGS demonstrated the ongoing dissemination of four main high-risk clones: ST13, ST17, ST147, and ST307, while no clones belonging to the European predominant clonal groups (CG15 and CG258) were found. Moreover, we describe one K. pneumoniae ST39-KL62 that coproduced the NDM-1 carbapenemase and the extended-spectrum beta-lactamase CTX-M-15, and one K. pneumoniae ST29-KL54 GES-5 and BEL-1 coproducer. Furthermore, a high prevalence of iron siderophores were present in all CRK strains, with several strains presenting both colibactin and the hypermucoviscosity phenotype. Thus, the data presented here highlight an uncommon molecular epidemiology pattern in Portugal when compared with most European countries, further supporting the emergence and dissemination of nonclonal group 258 hypervirulent multidrug high-risk clones and the need to promote in-depth hospital molecular surveillance studies.