Editorial

Publisher Copyright: © 2022 The AuthorsEvery two years, the Pollutant Toxic Ions and Molecules Conference, PTIM, meets the environmentalist, biologist, chemists and health researchers in Costa de Caparica, Portugal, to showcase the latest technologies, methodologies and research advances in pollution detection, contamination control, remediation, and related health issues, as well as policy implications.publishersversionpublishe

Multilocus Sequence Typing for characterization of potential risk ESBLs-producing Escherichia coli isolated from pigs, including strains of new singletons ST2528, ST2524 and ST2525

Infections caused by Escherichia coli harboring extended-spectrum beta-lactamases (ESBL) have a tremendous impact on public health, because of treatment complications. ESBL-producing E. coli are increasingly reported in healthy food-producing animals that can spread to humans either by direct contact or, more importantly, through the food chain. Here we describe a molecular survey aimed at determining the population structure and dynamics of ESBL-producing E. coli strains recovered from healthy pigs slaughtered for human consumption in Portugal. For this purpose, a total of 71 faecal samples from pigs were collected (2008 to 2009) in different geographical regions of Portugal. Susceptibility to 16 antibiotics was tested by disk-diffusion method in all recovered isolates and ESBL detection was carried out by double-disk test. PCR and sequencing methods characterized blaESBL genes responsible for the ESBL-phenotype. In addition, we used multilocus sequence typing (MLST) to identify the genetic lineages of all ESBL-producing E. coli strains, which were characterized by sequencing the internal fragments of 7 housekeeping genes (adk, fumC, gyrB, icd, mdh, purA, recA); the MLST database was used to determine allelic profiles and for sequence type (ST) and clonal complex (CC) assignment. Among the 35 ESBL-producing strains, MLST analysis revealed 9 different STs under 6 CCs and 9 singletons STs. The CC10 and CC155 were the most common CCs, with 4 and 11 isolates, respectively. Two other isolates were assigned to the CC101. Moreover, 5 strains were included in 3 new STs; 3 of them were identified in a new allele for the fumC gene that originated the new ST2528; in addition, 2 isolates were registered as ST2524 and ST2525 through new combination of alleles. Through the MLST database we found that ST656 (CC10) and ST8 (CC165) have a higher homology to ST2524 and ST2525, respectively. However, by the definition of CCs, ST2524 and ST2525 most likely belong to CC10 and CC165, respectively. Our data shows the presence of ESBL producing E. coli isolates in pigs slaughtered for human consumption and raises important questions in the potential risk factors to public health due to the transmission of bacteria carrying resistance through the food chain, and spreading resistance to other bacteria of human clinical significance. A great heterogeneity of MLST types was observed, among which CC10, CC155 and CC101 have already been associated with human clinical isolates

Multilocus Sequence Typing of Vancomycin-Resistant Enterococcus faecium isolated from pigs in Portugal

Vancomycin-resistant enterococci (VRE) first appeared in the late 1980s in a few European countries. In the last two decades, however, vancomycin-resistant Enterococcus faecium (VREfm) as became an emergent and challenging nosocomial problem. Specific clonal groups of E. faecium show an enhanced capacity to disseminate in the nosocomial setting. These strains can be assigned to distinct clonal groups or complexes based on DNA sequence-based typing (multi-locus sequence typing - MLST). In this context, we used the MLST technic to study the clonal relatedness of 18 VREfm strains previously isolated from pigs at slaughter level, in Portugal. These strains have been phenotypic and genotypic characterized in a previous study (1). For this purpose, internal 400–600-bp fragments of housekeeping genes were amplified and sequenced: adk, atpA, ddl, gdh, gyd, purK and pst (2). The sequences obtained were analysed and compared against the http://mlst.ucc.ie/ database. The combination of the seven obtained alleles, for each isolate, allows us to determine the corresponding sequence type (ST) and clonal complex (CC). MLST analyses revealed sequence type 5 (ST5) (n =5) and ST139 (n=12). These E. faecium sequence types belong to clonal complex 5 (CC5). Although ST139 is farthest from ST5, from which differs in three alleles, it also belongs to CC5. Strains belonging to CC5 are recognized to be circulating among European pigs. Although E. faecium CC5 are commonly found among animals they have also been isolated from humans. Furthermore, four of the isolates assigned to ST5 showed high-level resistance (HLR) to kanamycin and streptomycin, what can be of concern. In case of severe enterococcal infections the synergistic and bactericidal therapy can be reliably achieved with the addition of an aminoglycoside to β-lactamic antibiotics (or other cell wall agent such as vancomycin), as long as the organism does not exhibit HLR to the aminoglycoside. The recovery of E. faecium CC5 clone from slaughtered animals is of concern, since these strains may have the ability to either colonize humans or cause human infections

A Espectrometria de Massa de Alta Resolução na Medicina Personalizada: Proteína 4 de Ligação ao Retinol como Candidata a Biomarcador Preditor de Progressão no Carcinoma Urotelial da Bexiga

Introduction: Bladder cancer (BC) is the seventh most commonly diagnosed cancer in males. A large proportion of T1 cases and some Ta cases are under-staged and the significant risk of residual tumour after initial TURB of TaT1 lesions has been demonstrated. A second TURB is recommended in T1 tumours because it can increase recurrence-free survival (RFS) providing prognostic information. Non-invasive methods differentiating bladder cancer stages would be essential for diagnosis of under staged cases. Previously we demonstrated that a panel of urinary proteins measured by high resolution mass spectrometry could predict bladder cancer stages namely Ta, T1 and T2 cases. Our aim is to increase the accuracy of the biomarker panel for BC stage differentiation using a more patients, also intending to identify proteins that could be a signature to bladder cancer progression. Methods: Forty-eight urine samples were collected from volun- teers of these groups: 20 patients with bladder cancer stage Ta; 19 patients with BC stage T1 and 9 patients with BC stage T2+ (T2, T3 and T4). Urinary proteome was cleaned and digested using the Filter-Aided Sample Preparation methodology and analysed by liquid chromatography-mass spectrometry. For protein identification and label-free quantification we used MaxQuant, the data was further interrogated with the bioinformatics platform Perseus. Results: A biomarker panel was developed which consists of 87 proteins which were down-regulated between three different urothelial bladder carcinoma stages evaluated. Retinol-binding protein 4 (RBP4) consistently increases from Ta to T1, to T2+ (p <0.001) and in high grade tumours (p = 0.006). Conclusion: Our results showed a proteomic biomarker panel capable to differentiate bladder cancer stages. Besides, retinolbinding protein 4 can be a candidate signature of progression.Introdução: O carcinoma urotelial da bexiga é a sétima neoplasia mais comum dos homens. Uma proporção significativa de T1 e Ta são subestadiados e o risco de doença residual já foi demonstrado. Assim, a segunda RTU-V recomenda-se nos tumores T1 e está associada ao aumento da taxa livre de recidiva fornecendo informação prognóstica. Por isso, métodos não invasivos, capazes de diferenciar os estadios assim como diagnosticar os casos subestadiados são cada vez mais necessários. Num estudo prévio, desenvolvemos um painel de proteínas identificadas por espetrometria de massa de alta resolução capaz de diferencias os estadios Ta, T1 e T2+ (T2 a T4). O objetivo deste estudo consiste em aumentar a eficácia deste painel em diferenciar os estadios do carcinoma urotelial da bexiga e associadamente identificar candidatos promissores a biomarcadores de progressão. Métodos: Colheram-se 48 urinas de voluntários dos seguintes grupos: 20 doentes com carcinoma urotelial da bexiga Ta; 19 doentes com T1 e 9 doentes com T2 + (T2, T3 e T4). O proteoma urinário foi preparado usando a metodologia filter-aided sample e analisado através da cromotografia liquida e espetrometria de massa de alta resolução. Para a identificação e quantificação das proteínas utilizamos o MaxQuant e os resultados analisados pela plataforma bioinformática Perseus. Resultados: Um painel de biomarcadores proteicos com 87 proteinas sobre e sub expressas nos diferentes estadios foi conseguido. A proteína de ligação ao retinol 4 aumentou consistentemente de Ta para T1 e T2+ (p < 0,001) e nos tumores de alto grau comparativamente aos de baixo grau (p = 0,006). Conclusão: O estudo mostrou um painel de biomarcadores proteicos capaz de diferenciar os estadios do carcinoma da bexiga. Além disso, a proteína de ligação ao retinol 4 poderá ser um candidato promissor a biomarcador de progressão.

Biochemical network analysis of protein-protein interactions to follow-up T1 bladder cancer patients

PM003/2016). LBC, JLC, CL, RB, and HMS acknowledge the funding provided by the Associate Laboratory for Green Chemistry LAQV which is financed by national funds from FCT/MCTES, Fundação para a Ciência e Tecnologia and Ministério da Ciência, Tecnologia e Ensino Superior, through the projects UIDB/50006/2020 and UIDP/50006/2020. HMS acknowledges the Associate Laboratory for Green Chemistry-LAQV (LA/P/0008/2020) funded by FCT/MCTES for his research contract. LBC thanks the FCT/MCTES for his Ph.D. grant (SFRH/BD/144222/2019). The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE [30] partner repository with the data set number PXD026784. Publisher Copyright: © 2023 The AuthorsBladder cancer (BCa) is a prevalent disease with a high risk of aggressive recurrence in T1-stage patients. Despite the efforts to anticipate recurrence, a reliable method has yet to be developed. In this work, we employed high-resolution mass spectrometry to compare the urinary proteome of T1-stage BCa patients with recurring versus non-recurring disease to uncover actionable clinical information predicting recurrence. All patients were diagnosed with T1-stage bladder cancer between the ages of 51 and 91, and urine samples were collected before medical intervention. Our results suggest that the urinary myeloperoxidase to cubilin ratio could be used as a new tool for predicting recurrence and that dysregulation of the inflammatory and immune systems may be a key driver of disease worsening. Furthermore, we identified neutrophil degranulation and neutrophil extracellular traps (NETs) as key pathways in the progression of T1-stage BCa. We propose that proteomics follow-up of the inflammatory and immune systems may be useful for monitoring the effectiveness of therapy. Significance: This article describes how proteomics can be used to characterize tumor aggressiveness in patients with the same diagnosis of bladder cancer (BCa). LC-MS/MS in combination with label free quantification (LFQ) were used to explore potential protein and pathway level changes related to the aggressiveness of the disease in 13 and 17 recurring and non-recurring T1 stage BCa patients. We have shown that the MPO/CUBN protein ratio is a candidate for a urine prognosis tool in BCa. Furthermore, we identify dysregulation of inflammation process as a driver for BCa recurrence and progression. Moreover, we propose using proteomics to track the effectiveness of therapy in the inflammatory and immune systems.publishersversionpublishe

High efficacy of ozonated oils on the removal of biofilms produced by methicillin-resistant staphylococcus aureus (MRSA) from infected diabetic foot ulcers

© 2020 by the authors. Ozone has a high wound healing capacity and antibacterial properties and can be used as a complementary treatment in infections. Methicillin-resistant S. aureus (MRSA) is the most common pathogen found in infected diabetic foot ulcers. Most of MRSA are resistant to several classes of antibiotics and, therefore, there is a need for new, effective, and well-tolerated agents. Thus, we aimed evaluate the antimicrobial and antibiofilm potentials of ozonated vegetable oils against MRSA strains isolated from diabetic foot ulcers. Six ozonated oils were produced with concentrations of ozone ranging from 0.53 to 17 mg of ozone/g of oil. The peroxide values were determined for each oil. Ozonated oils content on fatty acid was determined by gas chromatography equipped with a flame ionization detector. The antimicrobial susceptibility testing was performed by the Kirby-Bauer disk diffusion method and the effect of ozonated oils on biofilm formation ability and on established biofilms was investigated. In general, the content in identified unsaturated fatty acid in oils decreased with the increase of ozonation time and, consequently, the peroxide value increased. Most bacterial strains were inhibited by ozonated oil at a concentration of 4.24 mg/g. Ozonated oils had moderate to high ability to remove adhered cells and showed a high capacity to eradicate 24 h old biofilms. Our results show promising use of ozonated oils on the treatment of infections, in particular those caused by multidrug-resistant MRSA strains.This work was funded by the R&D Project CAREBIO2—Comparative assessment of antimicrobial resistance in environmental biofilms through proteomics—towards innovative theranostic biomarkers, with reference NORTE-01-0145-FEDER-030101 and PTDC/SAU-INF/30101/2017, financed by the European Regional Development Fund (ERDF) through the Northern Regional Operational Program (NORTE 2020) and the Foundation for Science and Technology (FCT). This work was supported by the Associate Laboratory for Green Chemistry—LAQV, which is financed by national funds from FCT/MCTES (UIDB/50006/2020). Joana S. Amaral is grateful to the Foundation for Science and Technology (FCT, Portugal) for financial support by national funds FCT/MCTES to CIMO (UIDB/00690/2020). Vanessa Silva is grateful to FCT (Fundação para a Ciência e a Tecnologia) for financial support through PhD grant SFRH/BD/137947/2018.info:eu-repo/semantics/publishedVersio

High frequency of the EMRSA-15 clone (ST22-MRSA-IV) in hospital wastewater

Hospital wastewaters often carry multidrug-resistant bacteria and priority pathogens, such as methicillin-resistant Staphylococcus aureus (MRSA). Pathogens and antibiotic resistance genes present in wastewaters may reach the natural environment facilitating their spread. Thus, we aimed to isolate MRSA from wastewater of 3 hospitals located in the north of Portugal and to characterize the isolates regarding the antimicrobial resistance and genetic lineages. A total of 96 wastewater samples were collected over six months. The water was filtered, and the filtration membrane was immersed in BHI broth supplemented with 6.5% of NaCl and incubated. The inoculum was streaked in ORSAB agar plates for MRSA isolation. The isolates susceptibility testing was performed against 14 antimicrobial agents. The presence of resistance and virulence genes was accessed by PCR. Molecular typing was performed in all isolates. From the 96 samples, 28 (29.2%) were MRSA-positive. Most isolates had a multidrug-resistant profile and carried the mecA, blaZ, aac(6′)-Ie-aph(2′′)-Ia, aph(3′)-IIIa, ermA, ermB, ermC, tetL, tetM, dfrA dfrG and catpC221 genes. Most of the isolates were ascribed to the immune evasion cluster (IEC) type B. The isolates belonged to ST22-IV, ST8-IV and ST105-II and spa-types t747, t1302, t19963, t6966, t020, t008 and tOur study shows that MRSA can be found over time in hospital wastewater. The wastewater treatment processes can reduce the MRSA load. The great majority of the isolates belonged to ST22 and spa-type t747 which suggests the fitness of these genetic lineages in hospital effluents.info:eu-repo/semantics/publishedVersio

Diversity, Antimicrobial Resistance and Clonal Lineages

This work was funded by the R&D Project CAREBIO2: Comparative assessment of antimicrobial resistance in environmental biofilms through proteomics—towards innovative thera-nostic biomarkers, with reference NORTE-01-0145-FEDER-030101. This work was supported by the Associate Laboratory for Green Chemistry-LAQV, which is financed by national funds from FCT/MCTES and by the UIDB/CVT/00772/ 2020 project funded by the Portuguese Foundation for Science and Technology (FCT). Publisher Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland.Owls are nocturnal predators that inhabit urbanized and farmlands. They are in direct contact with other animals, both livestock and small wild rodents that they mostly feed on. Staphylococci can be both commensal and pathogenic bacteria that are widespread across the various ecological niches. We aimed to isolate staphylococci from owls and to characterize their antimicrobial resistance, virulence factors and genetic lineages. Swab samples were collected from the throat and cloaca of 114 owls admitted to two rehabilitation centers in Portugal. The identification of staphylococci species was performed by MALDI-TOF. Staphylococci antimicrobial resistance and virulence genes were investigated by means of the disk diffusion method and PCR. Staphylococcus aureus isolates were characterized by MLST, agr and spa-typing. Of the tested animals, 66 isolates were recovered, including 10 different species of staphylococci, of which 25 were coagulase-positive (CoPS) and 41 were coagulase-negative (CoNS). Twenty-three S. aureus were isolated, of which one mecC-MRSA was identified. The isolates were mainly resistant to penicillin, aminoglycosides, clindamycin and tetracycline. mecC-MRSA belonged to ST1245 and spa-type t843 and the remaining S. aureus were ascribed to 12 STs and 15 spa types. A high diversity of clonal lineages was identified among the S. aureus isolated from wild owls. Owls feed mainly on small rodents often exposed to waste and anthropogenic sources, which may explain the moderate prevalence of S. aureus in these animals.publishersversionpublishe

A One Health Approach Molecular Analysis of Staphylococcus aureus Reveals Distinct Lineages in Isolates from Miranda Donkeys (Equus asinus) and Their Handlers

This article belongs to the Special Issue Antibiotics and Alternative Treatments in Zoonosis Therapy.Donkeys (Equus asinus) are in decline in Europe. Occupational exposure to farm animals has been associated with increased staphylococci carriage. We aimed to isolate S. aureus and coagulase-negative staphylococci (CoNS) from donkeys and handlers and characterize the antimicrobial resistance profiles and genetic lineages of S. aureus strains. Oral and nasal swab samples were collected from 49 Miranda donkeys and 23 handlers from 15 different farms. Staphylococci species were identified by MALDI-TOF MS. The presence of antimicrobial resistance genes and virulence factors was investigated by PCR. Molecular typing was performed in S. aureus isolates. From the 49 donkey samples, 4 S. aureus (8.2%) and 21 CoNS (42.9%) were isolated. Ten handlers (43.5%) were carriers of S. aureus and 4 (17.4%) carried CoNS. The CoNS isolates showed resistance to several classes of antimicrobials encoded by the mecA, aph (3')-IIIa, ant (4')-Ia, tetM, tetK, lnuA, ermB, ermC, dfrA and dfrG genes. S. aureus isolates were resistant to penicillin, aminoglicosides and tetracycline harboring the blaZ, aph (3')-IIIa, tetL, tetM and tetK genes. All S. aureus isolates from donkeys belonged to ST49 and spa-type t208 while the strains isolated from the handlers were ascribed to 3 STs and 7 spa-types. However, human isolates were from different STs than the donkey isolates. Donkeys are mainly colonized by methicillin-resistant S. sciuri. S. aureus transmission between donkeys and their handlers appears not to have occurred since the isolates belonged to different genetic lineages.This work was funded by the R&D Project CAREBIO2: Comparative assessment of antimicrobial resistance in environmental biofilms through proteomics—towards innovative theranostic biomarkers, with reference NORTE-01-0145-FEDER-030101 and PTDC/SAU-INF/30101/2017, financed by the European Regional Development Fund (ERDF) through the Northern Regional Operational Program (NORTE 2020) and the Foundation for Science and Technology (FCT). This work was supported by the Associate Laboratory for Green Chemistry-LAQV, which is financed by national funds from FCT/MCTES (UIDB/50006/2020 and UIDP/50006/2020) and by the projects UIDB/CVT/00772/2020 and LA/P/0059/2020 funded by the Portuguese Foundation for Science and Technology (FCT). Vanessa Silva is grateful to FCT (Fundacão para a Ciência e a Tecnologia) for financial support through the PhD grant SFRH/BD/137947/2018.info:eu-repo/semantics/publishedVersio

Genetic characterization of methicillin-resistant staphylococcus aureus isolates from human bloodstream infections: detection of mlsb resistance

In this study we aimed to characterize antimicrobial resistance in methicillin-resistant Staphylococcus aureus (MRSA) isolated from bloodstream infections as well as the associated genetic lineages of the isolates. Sixteen MRSA isolates were recovered from bacteremia samples from inpatients between 2016 and 2019. The antimicrobial susceptibility of these isolates was tested by the Kirby–Bauer disk diffusion method against 14 antimicrobial agents. To determine the macrolide–lincosamide–streptogramin B (MLSB) resistance phenotype of the isolates, erythromycin-resistant isolates were assessed by double-disk diffusion (D-test). The resistance and virulence genes were screened by polymerase chain reaction (PCR). All isolates were characterized by multilocus sequence typing (MLST), spa typing, staphylococcal chromosomal cassette mec (SCCmec) typing, and accessory gene regulator (agr) typing. Isolates showed resistance to cefoxitin, penicillin, ciprofloxacin, erythromycin, fusidic acid, clindamycin, and aminoglycosides, confirmed by the presence of the blaZ, ermA, ermC, mphC, msrA/B, aac(6’)-Ie-aph(2’’)-Ia, and ant(4’)-Ia genes. Three isolates were Panton–Valentine-leukocidin-positive. Most strains (n = 12) presented an inducible MLSB phenotype. The isolates were ascribed to eight spa-types (t747, t002, t020, t1084, t008, t10682, t18526, and t1370) and four MLSTs (ST22, ST5, ST105, and ST8). Overall, most (n = 12) MRSA isolates had a multidrug-resistance profile with inducible MLSB phenotypes and belonged to epidemic MRSA clones.info:eu-repo/semantics/publishedVersio