João Botelho

HPLC-PAD-atmospheric pressure chemical ionization-MS metabolite profiling of cytotoxic carotenoids from the echinoderm Marthasterias glacialis (spiny sea-star).

An HPLC-PAD-atmospheric pressure chemical ionization-MS metabolite profiling analysis was conducted on the marine echinoderm Marthasterias glacialis (spiny sea-star). Bio-guided purification of the methanolic extract led to the isolation of several carotenoids, namely zeaxanthin, astaxanthin and lutein. These compounds were characterized using both UV-Vis characteristics and MS spectra interpretation. No previous works addressed the MS analysis of carotenoids present in this organism. The purified carotenoid fraction displayed a strong cell proliferation inhibition against rat basophilic leukemia RBL-2H3 (IC(25)=268 microg/mL) cancer cell line. Against healthy V79 (rat lung fibroblasts (IC(25)=411 microg/mL)) cell line, however, toxicity was lower, as it is desired for anti-cancer molecules. This study suggests that M. glacialis may constitute a good source of bioactive compounds that can be used as lead compounds for the pharmaceutical industry.

Unsuitability of MALDI-TOF MS to discriminate Acinetobacter baumannii clones under routine experimental conditions.

MALDI-TOF MS (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry) is now in the forefront for routine bacterial species identification methodologies, being its value for clonality assessment controversial. In this work we evaluated the potential of MALDI-TOF MS for assisting infection control by depicting Acinetobacter baumannii clones. Mass spectra of 58 A. baumannii clinical isolates belonging to the worldwide spread lineages (ST98, ST103, ST208, and ST218) isolated in our country, were obtained and analyzed with several chemometric tools (pseudo gel views, peakfind function, and partial least squares discriminant analysis). The clonal lineages were obtained using the “Oxford” scheme, belonging ST98, ST208, and ST218 to the international clone II and ST103 to an epidemic clonal lineage (SG5). Additionally, mass spectra of a highly diverse international collection of 38 isolates belonging to 22 sequence types (STs) were obtained for further comparisons. Pseudo gel views and direct peak pattern analysis did not allow the discrimination of A. baumannii isolates belonging to ST98, ST103, ST208, or ST218. Moreover, a partial least square discriminant analysis of the mass spectra considering two spectral ranges (2–20 kDa and 4–10 kDa) revealed a poor degree of discrimination with only 64.6 and 65.8% of correct ST assignments, respectively. Also, mass spectra of the international isolates (n = 38, 22STs) revealed a very congruent peak pattern among them as well as among the four lineages included in this work. Despite the increasing interest of MALDI-TOF MS for bacterial typing at different taxonomical levels, we demonstrated, using routine experimental conditions, the unsuitability of this methodology for A. baumannii clonal discrimination.

Characterization of a new genetic environment associated with GES-6 carbapenemase from a Pseudomonas aeruginosa isolate belonging to the high-risk clone ST235

Sir, Pseudomonas aeruginosa is an opportunistic pathogen commonly associated with nosocomial infections. The spread of P. aeruginosa strains resistant to carbapenems constitutes an emerging threat worldwide. The GES family of enzymes has been identified in several Gram-negative bacteria, such as P. aeruginosa, Klebsiella pneumoniae and Escherichia coli, being responsible for an extended resistance spectrum to several b-lactams. To date, according to the Lahey Clinic web site (http://www.lahey.org/Studies/), 24 variants of GES have been identified with some presenting carbapenemase activity, which is a matter of great concern. Few GES variants have been identified in Portugal, all of them found in Enterobacteriaceae. In Portugal, carbapenemases associated with P. aeruginosa include VIM-2 (the most frequent) and also IMP-5. Here, we report the first known worldwide description of the blaGES-6 gene in P. aeruginosa strains associated with a new genetic environment and also the first description of GES-producing P. aeruginosa in Portugal. In June 2012, a carbapenem-resistant P. aeruginosa isolate was recovered from bronchial secretions of a patient with pneumonia admitted to the intensive care unit of a Portuguese hospital. Antibiotic susceptibility assessed by standard disc diffusion, agar dilution and Etest methods demonstrated that the isolate was XDR, being non-susceptible to imipenem (MIC .32 mg/L), meropenem (MIC .32 mg/L), ceftazidime, cefepime, aztreonam, piperacillin/tazobactam, gentamicin, netilmicin, amikacin, tobramycin and ciprofloxacin, but susceptible to colistin (MIC1⁄42 mg/L). Carbapenemase production was confirmed by UV spectrophotometric assay and Blue-Carba. Screening of bla genes encoding metallo-b-lactamases (MBLs) gave negative results. The occurrence of ESBLs (blaBEL, blaVEB, blaPER and blaGES) was searched by PCR, revealing the presence of blaGES, which by sequencing was identified as blaGES-6. The nucleotide sequence differed from blaGES-6 (GenBank accession no. AY494718) by only one nucleotide (G instead of A at position 54), corresponding to a silent mutation. GES-6 is a class A b-lactamase with carbapenemase activity, differing from GES-1 by two amino acid substitutions (E104K and G170S), that was only described previously in Greece in a K. pneumoniae clinical isolate with reduced susceptibility to carbapenems. Association of blaGES-6 with class I integrons was performed by PCR and sequencing. A new class I integron structure of 5000 bp named In1076 by INTEGRALL (http://integrall.bio.ua.pt/) revealed an array of two gene cassettes attached to an IS (Figure 1; GenBank accession no. KM210290), with the blaGES-6 gene in the first position. The start codon (ATG) of blaGES-6 was preceded by two putative promoter regions [with a strong promoter, Pc; 235 (TTGACA) and 210 (TAAACT)] and a P2 promoter in its inactive form. An aacA7 gene encoding a type I aminoglycoside acetyltransferase [AAC(6′)-II], which confers resistance to amikacin, netilmicin and tobramycin, was found immediately downstream of the blaGES-6 gene. The 59 base element of the aacA7 gene cassette was interrupted at position 13 by a novel IS of 1543 bp, belonging to the IS110 family, IS1111 group, named ISPa55 and deposited in the IS finder database (https://www-is.biotoul.fr/) (Figure 1). Members of this group do not originate direct flanking target repeats and frequently encode a single ORF flanked by relatively long non-coding sequences (ISPa55 presented 78 and 330 bp at the left and right ends, respectively). The subterminal inverted repeats (IRs) (typical of the IS1111 group) were 5′-GAGTAAAAAGGAGACTTCCCG-3′ (left IR) and 5′-CGGGAAGCTCCTTATG-3′ (right IR). ISPa55 encodes a transposase related to the IS110 family, sharing 64% amino acid identity with that of ISPa34 identified in P. aeruginosa (GenBank accession no. ADC38937). The N-terminal regions of transposases of this family and the PIV/Moov family of DNA recombinases share a similar DEDD motif (D11E55D94D97 in ISPa55), suggesting they may use similar catalytic mechanisms. The junction promoter (Pjunc) originated by circularization of ISPa55 [TTGACG (17 bp) TAAAAA] is similar to the consensus promoters for representative IS1111 family members and is expected to increase the expression of the transposase transcript. ISPa55 disrupts the attC site of the aacA7 gene, as occurs with other IS110 family members that can insert themselves by site-specific recombination, which may compromise the excision of the corresponding gene cassettes. The genetic location of the blaGES-6 gene was determined by I-CeuI-PFGE followed by hybridization with probes specific for blaGES-6 and 16S rRNA genes. Both probes hybridized in the same band, revealing that the genetic platform bearing blaGES-6 was chromosomally located, although blaGES genes have been more frequently associated with plasmids. MLST was performed according to the P. aeruginosa database (http://pubmlst.org/paeruginosa/), revealing that the isolate belonged to the worldwide-disseminated high-risk ST235 clone associated with multidrug resistance and disseminated in Portuguese hospitals (J. Botelho, F. Grosso, C. Sousa and L. Peixe, unpublished results). Interestingly, ST235 has been previously associated with GES-1 and GES-5 in Spain. In summary, we present the first known worldwide report of the blaGES-6 carbapenemase gene in P. aeruginosa belonging to

Metabolic and biological prospecting of Coreopsis tinctoria

Coreopsis tinctoria Nutt., Asteraceae, flowering tops infusion has been traditionally used in many countries to control hyperglycaemia. In this work we report for the first time fatty acids and volatile compounds in this species. Fifteen fatty acids and sixteen volatile compounds were determined by GC-ITMS, being saturated fatty acids and monoterpenes the main compounds. The antioxidant and antibacterial potential of this matrix was checked for the first time by several in vitro assays. A concentration-dependent activity was noticed against DPPH, nitric oxide and superoxide radicals. Antibacterial capacity was assessed against Gram-positive and Gram-negative bacteria, being more effective against the first. Additionally, acetylcholinesterase and butyrylcholinesterase inhibitory activity was also evaluated, but no effect was found. Our results provide evidence of a wide diversity of compounds with several biological properties, improving the knowledge on this poorly studied matrix, which can lead to an increment of the use of C. tinctoria flowering tops, namely in food and pharmaceutical applications.

The complete nucleotide sequence of an IncP-2 megaplasmid unveils a mosaic architecture comprising a putative novel blaVIM-2-harbouring transposon in Pseudomonas aeruginosa

Objectives In Pseudomonas aeruginosa , bla VIM-2 has been mostly associated with a chromosomal location and rarely with a plasmid backbone. Until now, only three complete bla VIM-2 -carrying plasmid sequences have been described in this species. Here we explore the modular structure of pJB37, the first bla VIM-2 -carrying megaplasmid described in P. aeruginosa . Methods The complete nucleotide sequence of plasmid pJB37 was determined with an Illumina HiSeq, with de novo assembly by SPAdes, annotation by RAST and searching for antimicrobial resistance genes and virulence factors. Conjugation assays were conducted. Results Megaplasmid pJB37 (464 804 bp long and GC content of 57.2%) comprised: an IncP-2 repA-oriV-parAB region; a conjugative transfer region ( traF , traG , virD2 and trbBCDEJLFGI genes); Tn 6356 , a new putative bla VIM-2 -carrying transposon; heavy metal (mercury and tellurite) resistance operons; and an arsenal of virulence genes. Plasmid pJB37 was transferable by conjugation to a spontaneous rifampicin-resistant mutant of P. aeruginosa PAO1. Here, a bla VIM-2 -harbouring In58 integron was associated with a new complex transposable structure, herein named Tn 6356 , suggesting that In58 was most likely acquired by insertion of this element. Conclusions The mosaic arrangement exhibited by the pJB37 IncP-2 megaplasmid, which highlights the vast assembly potential of distinct genetic elements in a Pseudomonas widespread plasmid platform, gives new insights into bacterial adaptation and evolution.

Characterization of the pJB12 Plasmid from Pseudomonas aeruginosa Reveals Tn6352, a Novel Putative Transposon Associated with Mobilization of the blaVIM-2-Harboring In58 Integron

ABSTRACT The blaVIM-2-carrying In58 integron has been linked to a chromosomal location in different bacterial species, including Pseudomonas aeruginosa. This work reports the first fully sequenced In58-harboring plasmid, which is significantly different from the two previously identified blaVIM-2-carrying plasmids in P. aeruginosa. blaVIM-2 might have been acquired by transposition of Tn6352, a novel transposon composed of the In58 and ISPa17 elements. The recognition of similar inverted repeat (IR) sites by ISPa17 reveals a common mobilization process associated with acquisition of the blaVIM-2 and blaVIM-1 genes.

Community-led comparative genomic and phenotypic analysis of the aquaculture pathogen Pseudomonas baetica a390T sequenced by Ion semiconductor and Nanopore technologies

&NA; Pseudomonas baetica strain a390T is the type strain of this recently described species and here we present its high‐contiguity draft genome. To celebrate the 16th International Conference on Pseudomonas, the genome of P. baetica strain a390T was sequenced using a unique combination of Ion Torrent semiconductor and Oxford Nanopore methods as part of a collaborative community‐led project. The use of high‐quality Ion Torrent sequences with long Nanopore reads gave rapid, high‐contiguity and ‐quality, 16‐contig genome sequence. Whole genome phylogenetic analysis places P. baetica within the P. koreensis clade of the P. fluorescens group. Comparison of the main genomic features of P. baetica with a variety of other Pseudomonas spp. suggests that it is a highly adaptable organism, typical of the genus. This strain was originally isolated from the liver of a diseased wedge sole fish, and genotypic and phenotypic analyses show that it is tolerant to osmotic stress and to oxytetracycline.

GES-14-Producing Acinetobacter baumannii Isolates in a Neonatal Intensive Care Unit in Tunisia Are Associated with a Typical Middle East Clone and a Transferable Plasmid

Aymen Mabrouk,a,b Filipa Grosso,c João Botelho,c Wafa Achour,b,d Assia Ben Hassen,b,d Luisa Peixec Université de Carthage, Faculté des Sciences de Bizerte, Tunis, Tunisiaa; Centre National de Greffe de Moelle Osseuse, Service des Laboratoires, Tunis, Tunisiab; UCIBIO-REQUIMTE, Laboratório de Microbiologia, Faculdade de Farmácia, Universidade do Porto, Porto, Portugalc; Université de Tunis El Manar, Faculté de Médecine de Tunis, Tunis, Tunisiad

Carbapenemases on the move: it′s good to be on ICE

The evolution and spread of antibiotic resistance is often mediated by mobile geneticelements. Integrative and conjugative elements (ICEs) are the most abundant conjugativeelements among prokaryotes. However, the contribution of ICEs to horizontal gene transferof antibiotic resistance has been largely unexplored. Here we report that ICEs belonging tomating-pair formation (MPF) classes G and T are highly prevalent among the opportunisticpathogen Pseudomonas aeruginosa, contributing to the spread of carbapenemase-encodinggenes (CEGs). Most CEGs of the MPFG class were encoded within class I integrons, which co-harbour genes conferring resistance to other antibiotics. The majority of the integrons werelocated within Tn3-like and composite transposons. A conserved attachment site could bepredicted for the MPFGclass ICEs. MPFTclass ICEs carried the CEGs within compositetransposons which were not associated with integrons. The data presented here provides aglobal snapshot of the different CEG-harbouring ICEs and sheds light on the underappreciatedcontribution of these elements for the evolution and dissemination of antibiotic resistanceon P. aeruginosa.

Two decades of blaVIM-2-producing Pseudomonas aeruginosa dissemination: an interplay between mobile genetic elements and successful clones

Objectives Information on clonal lineages and genetic platforms involved in the mobilization of carbapenemases between Pseudomonas aeruginosa strains in Portugal is scarce. Here, we outline the genetic drivers contributing to the occurrence of blaVIM-2-producing P. aeruginosa over two decades. Methods A collection of carbapenem-resistant P. aeruginosa clinical isolates (n = 263, 1995-2014) was screened for carbapenemase production by Blue-Carba and PCR. Antimicrobial susceptibility testing was performed according to EUCAST and clonal analysis by MLST. Nine isolates representing different integrons and STs were selected for WGS, followed by bioinformatics. Results Twenty-seven blaVIM-2-producing P. aeruginosa belonging to 10 STs were identified, with ST179 and ST111 being the most prevalent and persistent clones. blaVIM-2 was associated with seven class I integrons frequently co-harbouring aminoglycoside resistance genes. In58 was commonly identified, followed by derivatives and In100. blaVIM-2-harbouring transposons of the Tn3 and Tn402 families were linked to different plasmids or integrative conjugative elements of the clc family. Conclusions The dissemination of blaVIM-2 carrying integrons is associated with a complex interplay between different mobile genetic elements, including the overlooked integrative conjugative elements, and successful spread of particular clones.