Molecular characterization of bla(IMP-5), a new integron-borne metallo-beta-lactamase gene from an Acinetobacter baumannii nosocomial isolate in Portugal

Postbloom fruit drop (PFD) of citrus is caused by Colletotrichum acutatum. PFD isolates infect flower petals, induce abscission of small fruit and can cause severe yield loss on most citrus cultivars. Isolates from Key lime anthracnose (KLA) cause that disease on the Mexican lime, but also cause PFD on sweet orange. Both PFD and KLA isolates exhibited resistance to the common selection agents including hygromycin, bialaphos, benomyl and geneticin/G418. A genetic transformation system was developed for C. acutatum to confer resistance to sulfonylurea (chlorimuron ethyl) by expressing an acetolactate synthase gene (sur) cassette from Magnaporthe grisea. The protocol was tested on 11 different KLA and PFD isolates. The transformation frequencies were highly variable among isolates and among experiments (0-17.9 per microg circular DNA using 10(7) protoplasts). Southern blot analysis of transformants indicated that the plasmid vector was randomly integrated in multiple copies into the genome of C. acutatum. Addition of restriction enzymes or use of a vector with homologous sequences did not change the transformation frequencies, but tended to reduce the number integrated. Over 97% of the transformants retained the sulfonylurea resistance phenotype under non-selective conditions. Of 300 transformants tested, three were unable to cause necrotic lesions on detached Key lime leaves. The transformation method opens up opportunities for the genetic manipulation of C. acutatum.     

Use of morphometric parameters for tracking ovule and microspore evolution in grapevine (<Emphasis Type="Italic">Vitis vinifera</Emphasis> L., cv. “Aragonez”) and evaluation of their potential to improve <Emphasis Type="Italic">in vitro</Emphasis> somatic embryogenesis efficiency from gametophyte tissues

Somatic embryogenesis induction from in vitro cultured stamens and carpels is highly dependent on explants’ inoculation at specific developmental stages. To establish good correlations between measurable morphometric parameters of flowers or flower buds and developmental stages of micro- and macrosporogenesis, this procedure is the easiest way to simplify the in vitro culture procedures. These correlations were established here for the most important Iberian grapevine cultivar, the “Aragonez”, named “Tempranillo” in Spain and “Tinta Roriz” in the north of Portugal, and were based in floral buds and anther measurements. The anther length, with a correlation coefficient of 0.90, proved to be the best morphometric parameter to follow microsporogenesis evolution. A correlation between micro- and macrosporogenesis evolutionary stages was also positively established, allowing the use of morphometric parameters for tracking ovule evolution as well. Carpels in several evolutionary stages were in vitro cultured to evaluate the aging effect on the capacity for somatic embryogenesis induction. Explants inoculated in the earliest stages of macrosporogenesis presented the best results. Media culture formulations were also tested for ovary culture, with the best results being achieved with a 5:1 auxin/cytokinin ratio.     

Multiniche screening reveals the clinically relevant metallo-beta-lactamase VIM-2 in Pseudomonas aeruginosa far from the hospital setting: an ongoing dispersion process?

A screening study of the presence of metallo-beta-lactamases (IMP and VIM types and SPM-1) in isolates from different nonhospital sources was conducted, and it revealed the presence of bla(VIM-2), associated with the In58 class 1 integron, in two unrelated Pseudomonas aeruginosa strains from aquatic habitats. The results suggest that the hospital setting was the possible origin of these bla(VIM-2)-carrying strains.     

Beta-nitrostyrene derivatives as potential antibacterial agents: a structure-property-activity relationship study

A multidisciplinary project was developed, combining the synthesis of a series of beta-nitrostyrene derivatives and the determination of their physicochemical parameters (redox potentials, partition coefficients), to the evaluation of the corresponding antibacterial activity. A complete conformational analysis was also performed, in order to get relevant structural information. Subsequently, a structure-property-activity (SPAR) approach was applied, through linear regression analysis, aiming at obtaining a putative correlation between the physicochemical parameters of the compounds investigated and their antibacterial activity (both against standard strains and clinical isolates). The beta-nitrostyrene compounds displayed a lower activity towards all the tested bacteria relative to the beta-methyl-beta-nitrostyrene analogues. This was observed particularly for the 3-hydroxy-4-methoxy-beta-methyl-beta-nitrostyrene (IVb) against the Gram-positive bacteria (Staphylococcus aureus, Enterococcus faecalis and Enterococcus faecium). The SPAR results revealed the existence of a clear correlation between the redox potentials and the antibacterial activity of the series of beta-nitrostyrene derivatives under study.     

Identification of carbapenem‐resistant Acinetobacter baumannii clones using infrared spectroscopy

In this work we assessed the discriminatory ability of Fourier‐transform Infrared Spectroscopy (FTIR) in 22 representative isolates from a collection of 318 carbapenem‐hydrolyzing class D β ‐lactamases (CHDL)‐producing Acinetobacter spp. (5 hospitals; 2001–2008) previously characterized by DNA‐based typing methods. FTIR spectra were acquired with a Bruker spectrometer and analyzed with support of several chemometric tools. The results showed that FTIR spectroscopy was able to distinguish the main CHDL‐producing Acinetobacter baumannii lineages causing infection in Portugal, the ST103 carrying bla_OXA‐58, ST98 carrying bla_OXA‐24/40and ST92 carrying bla_OXA‐23. Moreover, this study revealed distinctive phenotypic features of A. baumannii lineages causing infections that might justify different epidemic potential. Spectroscopy may arise as a low cost and easily to perform alternative for typing A. baumannii isolates. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Differentiation of Bacillus pumilus and Bacillus safensis Using MALDI-TOF-MS

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) despite being increasingly used as a method for microbial identification, still present limitations in which concerns the differentiation of closely related species. Bacillus pumillus and Bacillus safensis, are species of biotechnological and pharmaceutical significance, difficult to differentiate by conventional methodologies. In this study, using a well-characterized collection of B. pumillus and B. safensis isolates, we demonstrated the suitability of MALDI-TOF-MS combined with chemometrics to accurately and rapidly identify them. Moreover, characteristic species-specific ion masses were tentatively assigned, using UniProtKB/Swiss-Prot and UniProtKB/TrEMBL databases and primary literature. Delineation of B. pumilus (ions at m/z 5271 and 6122) and B. safensis (ions at m/z 5288, 5568 and 6413) species were supported by a congruent characteristic protein pattern. Moreover, using a chemometric approach, the score plot created by partial least square discriminant analysis (PLSDA) of mass spectra demonstrated the presence of two individualized clusters, each one enclosing isolates belonging to a species-specific spectral group. The generated pool of species-specific proteins comprised mostly ribosomal and SASPs proteins. Therefore, in B. pumilus the specific ion at m/z 5271 was associated with a small acid-soluble spore protein (SASP O) or with 50S protein L35, whereas in B. safensis specific ions at m/z 5288 and 5568 were associated with SASP J and P, respectively, and an ion at m/z 6413 with 50S protein L32. Thus, the resulting unique protein profile combined with chemometric analysis, proved to be valuable tools for B. pumilus and B. safensis discrimination, allowing their reliable, reproducible and rapid identification.     

From farm to fork: Colistin voluntary withdrawal in Portuguese farms reflected in decreasing occurrence of mcr-1-carrying Enterobacteriaceae from chicken meat

Expansion of mcr-carrying Enterobacteriaceae (MCR-E) is a well-recognized problem affecting animals, humans and the environment. Ongoing global control actions involve colistin restrictions among food-animal production, but their impact on poultry-derived products is largely unknown, justifying comprehensive farm-to-fork studies. Occurrence of MCR-E among 53 chicken-meat batches supplied from 29 Portuguese farms shortly after colistin withdrawal was evaluated. Strains (FT-IR/MLST/WGS), mcr plasmids and their adaptive features were characterized by cultural, molecular and genomic approaches. We found high rates of chicken-meat batches (80%–100% – 4 months; 12% – the last month) with multiple MDR + mcr-1-carrying Escherichia coli (Ec-including ST117 and ST648-Cplx) and Klebsiella pneumoniae (Kp-ST147-O5:K35) clones, some of them persisting over time. The mcr-1 was located in the chromosome (Ec-ST297/16-farms) or dispersed IncX4 (Ec-ST602/ST6469/5-farms), IncHI2-ST2/ST4 (Ec-ST533/ST6469/5 farms and Kp-ST147/6-farms) or IncI2 (Ec-ST117/1-farm) plasmids. WGS revealed high load and diversity in virulence, antibiotic resistance and metal tolerance genes. This study supports colistin withdrawal potential efficacy in poultry production and highlights both poultry-production chain as a source of mcr-1 and the risk of foodborne transmission to poultry-meat consumers. Finally, in the antibiotic reduction/replacement context, other potential co-selective pressures (e.g., metals-Cu as feed additives) need to be further understood to guide concerted, effective and durable actions under 'One Health' perspective.     

Chromobacterium violaceum: Important Insights for Virulence and Biotechnological Potential by Exoproteomic Studies

Chromobacterium violaceum is a beta-proteobacterium with high biotechnological potential, found in tropical environments. This bacterium causes opportunistic infections in both humans and animals, that can spread throughout several tissues, quickly leading to the death of the host. Genomic studies identified potential mechanisms of pathogenicity but no further studies were done to confirm the expression of these systems. In this study 36 unique protein entries were identified in databank from a two-dimensional profile of C. violaceum secreted proteins. Chromobacterium violaceum exoproteomic preliminary studies confirmed the production of proteins identified as virulence factors (such as a collagenase, flagellum proteins, metallopeptidases, and toxins), allowing us to better understand its pathogenicity mechanisms. Biotechnologically interesting proteins (such as chitinase and chitosanase) were also identified among the secreted proteins, as well as proteins involved in the transport and capture of amino acids, carbohydrates, and oxidative stress protection. Overall, the secreted proteins identified provide us important insights on pathogenicity mechanisms, biotechnological potential, and environment adaptation of C. violaceum.