Browsing by Author "Melo-Cristino, J"
Now showing 1 - 10 of 13
Results Per Page
Sort Options
- Bombas de Efluxo em Pseudomonas Aeruginosa. Importância Clínica na TerapêuticaPublication . Caneiras, C; Marcos, C; Ferreira, T; Hanscheid, T; Melo-Cristino, J; Duarte, AAs infecções severas causadas por Pseudomonas Aeruginosa têm assumido na última década um papel de destaque, dado tratar-se de um microrganismo de difícil erradicação e com elevada resistência intrínseca e adquirida. Em P. aeruginosa foram caracterizados sete sistemas de bombas de efluxo, os quais contribuem para o desenvolvimento de fenótipos de multiresistência às classes de antibióticos clinicamente mais relevantes. Dado que a terapêutica seleccionada pode actuar como indutora e originar eventos mutacionais que promovem a hiperexpressão das bombas de efluxo, o tratamento destas infecções constitui um grande desafio terapêutico e impõe um conhecimento actualizado desta temática. Esta revisão foi assim desenvolvida com o intuito de rever a contribuição da hiperexpressão dos sistemas de bombas de efluxo em P.aeruginosa, na multirresistência aos antibióticos. Pretendeu-se também avaliar a possibilidade da utilização destes sistemas como alvos terapêuticos de modo a restabelecer a susceptibilidade aos antibióticos disponíveis.
- Clinical and Bacteriological Survey of Diabetic Foot Infections in LisbonPublication . Mendes, JJ; Marques-Costa, A; Vilela, C; Neves, J; Candeias, N; Cavaco-Silva, P; Melo-Cristino, JAIMS: An epidemiological survey of diabetic foot infections (DFIs) in Lisbon, stratifying the bacterial profile based on patient demographical data, diabetic foot characteristics (PEDIS classification), ulcer duration and antibiotic therapy. METHODS: A transversal observational multicenter study, with clinical data collection using a structured questionnaire and microbiological products (aspirates, biopsies or swabs collected using the Levine method) of clinically infected foot ulcers of patients with diabetes mellitus (DM). RESULTS: Forty-nine hospitalized and ambulatory patients were enrolled in this study, and 147 microbial isolates were cultured. Staphylococcus was the main genus identified, and methicillin-resistant Staphylococcus aureus (MRSA) was present in 24.5% of total cases. In the clinical samples collected from patients undergoing antibiotic therapy, 93% of the antibiotic regimens were considered inadequate based on the antibiotic susceptibility test results. The average duration of an ulcer with any isolated multi-drug resistant (MDR) organism was 29 days, and previous treatment with fluoroquinolones was statistically associated with multi-drug resistance. CONCLUSIONS: Staphylococcus aureus was the most common cause of DFIs in our area. Prevalence and precocity of MDR organisms, namely MRSA, were high and were probably related to previous indiscriminate antibiotic use. Clinicians should avoid fluoroquinolones and more frequently consider the use of empirical anti-MRSA therapy.
- Confronting Ceftolozane-Tazobactam Susceptibility in Multidrug-Resistant Enterobacterales Isolates and Whole-Genome Sequencing Results (STEP Study)Publication . Hernández-García, M; García-Fernández, S; García-Castillo, M; Melo-Cristino, J; Pinto, M; Gonçalves, E; Alves, V; Costa, E; Ramalheira, E; Sancho, L; Diogo, J; Ferreira, R; Silva, T; Chaves, C; Pássaro, L; Paixão, L; Romano, J; Cantón, J; STEP Study GroupCeftolozane-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.
- Decreasing Incidence and Changes in Serotype Distribution of Invasive Pneumococcal Disease in Persons Aged Under 18 Years Since Introduction of 10-Valent and 13-Valent Conjugate Vaccines in Portugal, July 2008 to June 2012Publication . Aguiar, SI; Brito, MJ; Horacio, AN; Lopes, JP; Ramirez, M; Melo-Cristino, JThe 10-valent pneumococcal conjugate vaccine (PCV10) became available in Portugal in mid-2009 and the 13-valent vaccine (PCV13) in early 2010. The incidence of invasive pneumococcal disease (IPD) in patients aged under 18 years decreased from 8.19 cases per 100,000 in 2008–09 to 4.52/100,000 in 2011–12. However, IPD incidence due to the serotypes included in the 7-valent conjugate vaccine (PCV7) in children aged under two years remained constant. This fall resulted from significant decreases in the number of cases due to: (i) the additional serotypes included in PCV10 and PCV13 (1, 5, 7F; from 37.6% to 20.6%), particularly serotype 1 in older children; and (ii) the additional serotypes included in PCV13 (3, 6A, 19A; from 31.6% to 16.2%), particularly serotype 19A in younger children. The decrease in serotype 19A before vaccination indicates that it was not triggered by PCV13 administration. The decrease of serotype 1 in all groups, concomitant with the introduction of PCV10, is also unlikely to have been triggered by vaccination, although PCVs may have intensified and supported these trends. PCV13 serotypes remain major causes of IPD, accounting for 63.2% of isolates recovered in Portugal in 2011–12, highlighting the potential role of enhanced vaccination in reducing paediatric IPD in Portugal.
- Dominance of Vaccine Serotypes in Pediatric Invasive Pneumococcal Infections in Portugal (2012-2015)Publication . Silva-Costa, C; Brito, MJ; Aguiar, SI; Lopes, JP; Ramirez, M; Melo-Cristino, JWe evaluated the impact of continued 13-valent pneumococcal conjugate vaccine (PCV13) use in the private market (uptake of 61%) in pediatric invasive pneumococcal disease (pIPD) in Portugal (2012-2015). The most frequently detected serotypes were: 3 (n = 32, 13.8%), 14 (n = 23, 9.9%), 1 (n = 23, 9.9%), 7F (n = 15, 6.4%), 19A (n = 13, 5.6%), 6B and 15B/C (both n = 12, 5.2%), and 24F, 10A and 12B (all with n = 10, 4.3%). Taken together, non-PCV13 serotypes were responsible for 42.2% of pIPD with a known serotype. The use of PCR to detect and serotype pneumococci in both pleural and cerebrospinal fluid samples contributed to 18.1% (n = 47) of all pIPD. Serotype 3 was mostly detected by PCR (n = 21/32, 65.6%) and resulted from a relevant number of vaccine failures. The incidence of pIPD varied in the different age groups but without a clear trend. There were no obvious declines of the incidence of pIPD due to serotypes included in any of the PCVs, and PCV13 serotypes still accounted for the majority of pIPD (57.8%). Our study indicates that a higher vaccination uptake may be necessary to realize the full benefits of PCVs, even after 15 years of moderate use, and highlights the importance of using molecular methods in pIPD surveillance, since these can lead to substantially increased case ascertainment and identification of particular serotypes as causes of pIPD.
- Imipenem-Relebactam Susceptibility in Enterobacterales Isolates Recovered from ICU Patients from Spain and Portugal (SUPERIOR and STEP Studies)Publication . Hernández-García, M; García-Castillo, M; Bou, G; Cercenado, E; Delgado-Valverde, M; Oliver, A; Pitart, C; Rodríguez-Lozano, J; Tormo, N; Melo-Cristino, J; Pinto, MF; Gonçalves, E; Alves, V; Vieira, AR; Ramalheira, E; Sancho, L; Diogo, J; Ferreira, R; Cruz, H; Chaves, C; Duarte, J; Pássaro, L; Díaz-Regañón, J; Cantón, RImipenem-relebactam is a novel β-lactam-β-lactamase inhibitor combination. We evaluated the in vitro activity of imipenem-relebactam and comparators against Enterobacterales clinical isolates recovered in 8 Spanish and 11 Portuguese intensive care units (ICUs) (SUPERIOR, 2016-2017; STEP, 2017-2018). Overall, 747 Enterobacterales isolates (378 Escherichia coli, 252 Klebsiella spp., 64 Enterobacter spp., and 53 other species) were prospectively collected from ICU patients with complicated intraabdominal (cIAI), complicated urinary tract (cUTI), and lower respiratory tract (LRTI) infections. MICs were determined (ISO-broth microdilution), and whole-genome sequencing (WGS) was performed in a subset of isolates displaying susceptible and resistant imipenem-relebactam MICs. Imipenem-relebactam (98.7% susceptible) showed similar activity to ceftazidime-avibactam (99.5% susceptible) and higher than ceftolozane-tazobactam (86.9% susceptible). Imipenem-relebactam was inactive against 1.3% (10/747) isolates, all of them due to carbapenemase production (9 K. pneumoniae and 1 E. cloacae). Imipenem-relebactam was active against 100% of extended-spectrum β-lactamase (ESBL)-E. coli and ESBL-Klebsiella spp. isolates and 80.4% of carbapenemase-Klebsiella spp. producers. Carbapenemase genes were confirmed by WGS in 41 Klebsiella spp.: OXA-48 (20/41), KPC-3 (14/41), OXA-181 (4/41), NDM-1 (1/41), OXA-48 + VIM-2 (1/41), and KPC-3 + VIM-2 (1/41). In Klebsiella spp. isolates, relebactam restored imipenem susceptibility in all KPC-3 producers, and resistant isolates (7/41) were mostly OXA-48 + CTX-M-15-K. pneumoniae high-risk clones (7/9). Intercountry differences were detected as follows: OXA-48 (17/21) was dominant in Spain, unlike KPC-3 (14/15) in Portugal. Imipenem-relebactam was 100% active against CTX-M-15-ST131-H30Rx-E. coli high-risk clone, predominant in both countries. Our results depict the potential role of imipenem-relebactam in ICU patients with cIAIs, cUTIs, and LRTIs due to wild-type ESBL- and carbapenemase-producing Enterobacterales, particularly KPC producers. IMPORTANCE We comparatively evaluate the in vitro activity of a drug combination consisting of a carbapenem (imipenem) and a novel inhibitor of beta-lactamases (relebactam), a mechanism that destroys beta-lactam antibiotics. We assess the activity against a collection of Enterobacterales clinical isolates recovered from difficult-to-treat infections in patients admitted to different intensive care units in Portugal and Spain. Imipenem-relebactam shows excellent activity in avoiding common resistance mechanisms in this setting, such as extended-spectrum beta-lactamases and carbapenemases widely distributed, including KPCs. We show few resistant isolates (<2%). Molecular characterization by whole-genome sequencing shows that most of the resistant isolates produced specific carbapenemase, such as OXA-48 or metalo-betalactamases. Our study updates the activity of imipenem-relebactam in light of current epidemiology in a hospital setting in which the use of this combination is needed due to the presence of infections due to multidrug-resistant isolates.
- 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 StudyPublication . García-Fernández, S; García-Castillo, M; Melo-Cristino, J; Pinto, M; Gonçalves, E; Alves, V; Vieira, AR; Ramalheira, E; Sancho, L; Diogo, J; Ferreira, R; Silva, D; Chaves, C; Pássaro, L; Paixão, LThe 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.
- In Vitro Design of a Novel Lytic Bacteriophage Cocktail with Therapeutic Potential Against Organisms Causing Diabetic Foot InfectionsPublication . Mendes, JJ; Leandro, C; Mottola, C; Barbosa, R; Silva, F; Oliveira, M; Vilela, C; Melo-Cristino, J; Górski, A; Pimentel, M; São-José, C; Cavaco-Silva, P; Garcia, MIn 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.
- Molecular Typing, Virulence Traits and Antimicrobial Resistance of Diabetic Foot StaphylococciPublication . Mottola, C; Semedo-Lemsaddek, T; Mendes, JJ; Melo-Cristino, J; Tavares, L; Cavaco-Silva, P; Oliveira, MDiabetes mellitus is a major chronic disease that continues to increase significantly. One of the most important and costly complications of diabetes are foot infections that may be colonized by pathogenic and antimicrobial resistant bacteria, harboring several virulence factors, that could impair its successful treatment. Staphylococcus aureus is one of the most prevalent isolate in diabetic foot infections, together with aerobes and anaerobes.
- A Phylogenomic Approach for the Analysis of Colistin Resistance-Associated Genes in Klebsiella Pneumoniae, its Mutational Diversity and Implications for Phenotypic ResistancePublication . Elias, R; Spadar, A; Phelan, J; Melo-Cristino, J; Lito, L; Pinto, M; Gonçalves, L; Campino, S; Clark, TG; Duarte, A; Perdigão, JThe 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 (mgrBIle13fs, pmrBGly207Asp, phoQHis339Asp and ramAIle28Met) 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.