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- Cytotoxic and Genotoxic Effects of Environmental Relevant Concentrations of Bisphenol A and Interactions with DoxorubicinPublication . Ramos, C; Ladeira, C; Zeferino, S; Dias, A; Faria, I; Cristovam, E; Gomes, M; Ribeiro, EBisphenol A (BPA) is one of the most widely utilized endocrine disruptors to which humans are exposed, particularity through ingestion. BPA is an aneugenic compound with a putative association to tumorigenesis. Although extensively studied in estrogen responsive cells, information regarding its effects on cells from the upper gastrointestinal tract exposed to free/active forms of BPA is still scarce. Similarly, BPA interactions with other drugs have been neglected, although it has been suggested to have a potential role in doxorubicin (DOX) chemoresistance. This study is intended to assess potential cytotoxic and genotoxic effects of BPA, as well as its interactions with DOX, in Human epithelial type 2 cells (Hep-2) originated from a human laryngeal carcinoma and in a DNA damage responsive cell line, the human lung fibroblasts (MRC-5). Cell viability was analyzed through the resazurin assay. The G protein-coupled estrogen receptor 1 (GPER) expression was visualized by immunodetection. Genotoxicity, namely DNA damage and oxidative DNA damage, were assessed by comet assay and micronuclei induction, and mitotic disruption was evaluated cytologically by fluorescent microscopy with DAPI staining. Cytotoxicity analysis showed that exposure to BPA per se does not affect cellular viability. Nevertheless, the genotoxic analysis showed that BPA induced an increase of DNA damage in the Hep-2 cell line and in oxidative damage in the MRC-5 cell line. An increase of micronuclei was also observed in both cell lines following BPA exposure. BPA and DOX co-exposures suggested that BPA acts as an antagonist of DOX effects in both cell lines. The interaction with DOX appears to be cell type dependent, exhibiting a non-monotonic response curve in MRC-5 cells, a GPER expressing cell line. Our study emphasizes the need for a deeper knowledge of BPA interactions, particularly with chemotherapeutic agents, in the context of risk assessment and public health.
- Lymphogranuloma Venereum: a Retrospective Analysis of an Emerging Sexually Transmitted Disease in a Lisbon Tertiary CenterPublication . Neves, JM; Ramos Pinheiro, R; Côrte‐Real, R; Borrego, MJ; Rodrigues, A; Fernandes, CBackground: Lymphogranuloma venereum (LGV) is a sexual transmitted infection (STI), currently endemic within the population of men who have sex with men (MSM) of Western Countries. L2B variant has been reported as the predominant strain in the current LGV epidemics, although a shift towards L2-434 has been observed in some European countries. Objectives: To evaluate and characterize the population with LGV infection diagnosed in Centro Hospitalar Universitário de Lisboa Central, Lisbon, Portugal. Methods: A retrospective analysis of all LGV diagnoses between 2016 and 2019 was performed. The diagnosis was established through ompA-genotyping of samples yielding a positive result to Chlamydia trachomatis (CT). All considered samples were retrieved from the clinician activity, through swabbing and urine analysis and CT infection diagnosis was obtained using real-time PCR. Results: During the period studied 16 279 CT diagnostics tests were employed, with a striking increase from 2016 (n = 467) to 2019 (n = 9362). A total of 1602 diagnoses of CT were established, from which 168 (10.5%) corresponded to LGV, with both infections showing a rising evolution, between 2016 and 2019, of 2.9 and 2.7 times, respectively. The majority of the LGV strains were genotyped as L2/434 (67.3%; n = 113). LGV predominantly affected MSM and men who have sex with men and women (97.0%; n = 163). Anorectal infection was the most prevalent one (90.5%; n = 152), being proctitis the main clinical presentation (76.2%; n = 128). Absence of symptoms was reported in almost 15% of the cases (n = 24). The presence of concomitant infection with human immunodeficiency virus was dominant (73.2%; n = 123) and the prevalence of one or more STI co-infections was about 60.1% (n = 99). Conclusions: An increasing evolution of CT and LGV testing and diagnosing was observable throughout the studied period. Characteristics of the population are similar with those described within LGV epidemics. In accordance with recent European studies, predominance towards L2 genotype was identified.
- Disclosing Azole Resistance Mechanisms in Resistant Candida Glabrata Strains Encoding Wild-Type or Gain-of-Function CgPDR1 Alleles Through Comparative Genomics and TranscriptomicsPublication . Salazar, SB; Pinheiro, MJ; Sotti-Novais, D; Soares, AR; Lopes, MM; Ferreira, T; Rodrigues, V; Fernandes, F; Mira, NPThe pathogenic yeast Candida glabrata is intrinsically resilient to azoles and rapidly acquires resistance to these antifungals, in vitro and in vivo. In most cases azole-resistant C. glabrata clinical strains encode hyperactive CgPdr1 variants, however, resistant strains encoding wild-type CgPDR1 alleles have also been isolated, although remaining to be disclosed the underlying resistance mechanism. In this study, we scrutinized the mechanisms underlying resistance to azoles of 8 resistant clinical C. glabrata strains, identified along the course of epidemiological surveys undertaken in Portugal. Seven of the strains were found to encode CgPdr1 gain-of-function variants (I392M, E555K, G558C, and I803T) with the substitutions I392M and I803T being herein characterized as hyper-activating mutations for the first time. While cells expressing the wild-type CgPDR1 allele required the mediator subunit Gal11A to enhance tolerance to fluconazole, this was dispensable for cells expressing the I803T variant indicating that the CgPdr1 interactome is shaped by different gain-of-function substitutions. Genomic and transcriptomic profiling of the sole azole-resistant C. glabrata isolate encoding a wild-type CgPDR1 allele (ISTB218) revealed that under fluconazole stress this strain over-expresses various genes described to provide protection against this antifungal, while also showing reduced expression of genes described to increase sensitivity to these drugs. The overall role in driving the azole-resistance phenotype of the ISTB218 C. glabrata isolate played by these changes in the transcriptome and genome of the ISTB218 isolate are discussed shedding light into mechanisms of resistance that go beyond the CgPdr1-signalling pathway and that may alone, or in combination, pave the way for the acquisition of resistance to azoles in vivo.
- Adrenal Vein Sampling in the Management of Primary Aldosteronism: The Added Value of Intraprocedural Cortisol AssessmentPublication . Manique, I; Amaral, S; Matias, A; Bouça, B; Serranito, S; Torres, J; Gutu, O; Bilhim, T; Coimbra, E; Rodrigues, I; Godinho, C; Cortez, L; Silva-Nunes, JIntroduction: Primary aldosteronism is the most common cause of secondary hypertension. Adrenal vein sampling is the gold standard for subtyping primary aldosteronism. However, this procedure is technically challenging and often has a low success rate. Our center is one of the very few performing this technique in our country with an increasing experience. Objective: The aim of this study was to evaluate the role of the cortisol intraprocedural assay in improving the performance of adrenal vein sampling. Design: We enrolled all of the patients with primary aldosteronism that underwent adrenal vein sampling from February 2016 to April 2023. The cortisol intraprocedural assay was introduced in October 2021. Methods: We enrolled a total of 50 adrenal vein samplings performed on 43 patients with the diagnosis of primary aldosteronism. In this sample, 19 patients and 24 patients underwent adrenal vein sampling before and after intraprocedural cortisol measurement, respectively. The procedure was repeated in seven patients (one before and six after intraprocedural cortisol measurement), given the unsuccess of the first exam. Selectivity of the adrenal vein sampling was assumed if the serum cortisol concentration from the adrenal vein was at least five times higher than that of the inferior vena cava. Lateralization was assumed if the aldosterone to cortisol ratio of one adrenal vein was at least four times the aldosterone to cortisol ratio of the contralateral side. Results: The mean age of the patients that underwent adrenal vein sampling (N = 43) was 55.2 ± 8.9 years, and 53.5% (n = 23) were female. The mean interval between the diagnosis of hypertension and the diagnosis of primary aldosteronism was 9.8 years (±9.9). At diagnosis, 62.8% of the patients (n = 27) had hypokalemia (mean value of 3 mmol/L (±0.34)), 88.4% (n = 38) had adrenal abnormalities on preprocedural CT scan, and 67.4% (n = 29) described as unilateral nodules. There were no statistically significant differences in the patients' baseline characteristics between the two groups (before and after intraprocedural cortisol measurement). Before intraprocedural cortisol measurement, adrenal vein sampling selectivity was achieved in 35% (n = 7) patients. Selectivity increased to 100% (30/30) after intraprocedural cortisol measurement (p < 0.001). With the exception of one patient who refused it, all patients with lateralized disease underwent unilateral adrenalectomy with normalization of the aldosterone to renin ratio postoperatively. Conclusions: The lack of effective alternatives in subtyping primary aldosteronism highlights the need to improve the success rate of adrenal vein sampling. In this study, intraprocedural cortisol measurement allowed a selectivity of 100%. Its addition to this procedure protocol should be considered, especially in centers with a low success rate.
- An Eleven-Year-Old Boy With Arthritis of the Hip Joint, and an 8-Year-Old Boy With SacroiliitisPublication . Mendo, T; Norte, S; Alves, P; Tavares, D; Pinto, M; Gouveia, C
- Gastrointestinal: Diarrhea and Bowel Thickening in Elderly Patients: a Diagnosis to Keep in MindPublication . Gamelas, V; Saraiva, R; Duarte Santos, J; Borges, V; Saiote, J; Costa Simões, J
- Epigenetic and Drug Response Modulation of Epigalocaten-In-3-Gallate in Staphylococcus aureus with Divergent Resistance PhenotypesPublication . Mira, AR; Zeferino, AS; Inácio, R; Delgadinho, M; Brito, M; Calado, CRC.; Ribeiro, EHealthcare-associated methicillin-resistant Staphylococcus aureus infections represent extremely high morbidity and mortality rates worldwide. We aimed to assess the antimicrobial potential and synergistic effect between Epigalocatenin-3-gallate (EGCG) and different antibiotics in S. aureus strains with divergent resistance phenotypes. EGCG exposure effects in epigenetic and drug resistance key modulators were also evaluated. S. aureus strains (n = 32) were isolated from infected patients in a Lisbon hospital. The identification of the S. aureus resistance phenotype was performed through automatized methods. The antibiotic synergistic assay was performed through disk diffusion according to EUCAST guidelines with co-exposure to EGCG (250, 100, 50 and 25 µg/mL). The bacteria's molecular profile was assessed through FTIR spectroscopy. The transcriptional expression of OrfX, SpdC and WalKR was performed by using qRT-PCR. FTIR-spectroscopy analysis enabled the clear discrimination of MRSA/MSSA strains and the EGCG exposure effect in the bacteria's molecular profiles. Divergent resistant phenotypes were associated with divergent transcriptional expression of the epigenetic modulator OrfX, particularly in MRSA strains, as well as the key drug response modulators SpdC and WalKR. These results clearly demonstrate that EGCG exposure alters the expression patterns of key epigenetic and drug response genes with associated divergent-resistant profiles, which supports its potential for antimicrobial treatment and/or therapeutic adjuvant against antibiotic-resistant microorganisms.
- Invasive and Subcutaneous Infections Caused by Filamentous Fungi: Report from a Portuguese Multicentric Surveillance ProgramPublication . Veríssimo, C; Toscano, C; Ferreira, T; Abreu, G; Simões, H; Diogo, J; Carvalho, D; Santiago, F; Lima, A; Queirós, AM; Sabino, RInvasive fungal infections (IFI) have significantly increased over the past years due to advances in medical care for the at-risk immunocompromised population. IFI are often difficult to diagnose and manage, and can be associated with substantial morbidity and mortality. This study aims to contribute to understanding the etiology of invasive and subcutaneous fungal infections, their associated risk factors, and to perceive the outcome of patients who developed invasive disease, raising awareness of these infections at a local level but also in a global context. A laboratory surveillance approach was conducted over a seven-year period and included: (i) cases of invasive and subcutaneous fungal infections caused by filamentous/dimorphic fungi, confirmed by either microscopy or positive culture from sterile samples, (ii) cases diagnosed as probable IFI according to the criteria established by EORTC/MSG when duly substantiated. Fourteen Portuguese laboratories were enrolled. Cases included in this study were classified according to the new consensus definitions of invasive fungal diseases (IFD) published in 2020 as follows: proven IFI (N = 31), subcutaneous fungal infection (N = 23). Those proven deep fungal infections (N = 54) totalized 71.1% of the total cases, whereas 28.9% were classified as probable IFI (N = 22). It was possible to identify the etiological fungal agent in 73 cases (96%). Aspergillus was the most frequent genera detected, but endemic dimorphic fungi represented 14.47% (N = 11) of the total cases. Despite the small number of cases, a high diversity of species were involved in deep fungal infections. This fact has implications for clinical and laboratory diagnosis, and on the therapeutic management of these infections, since different species, even within the same genus, can present diverse patterns of susceptibility to antifungals.
- 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.
- 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.