Catarina Marinho

Dissemination of antibiotic resistant Enterococcus spp. and Escherichia coli from wild birds of Azores Archipelago

Antibiotic-resistant is profoundly important to human and animal health, but the environmental reservoirs of resistance determinants are poorly understood, in particular in wild environments in remote Archipelagos. Moreover, the prevalence of antibiotic-resistant bacteria in wild bird populations is rather unknown. In the present study, we used the Azores Islands as a model study for antimicrobial resistance in a remote Archipelago, and examined the antibiotic resistance profile in enterococci and Escherichia coli recovered from faecal samples of wild birds collected in this Archipelago. A total of 138 enterococci and 115 E. coli isolates were analyzed for resistance to antimicrobial agents. Of the enterococcal isolates, Enterococcus faecalis was the most prevalent detected species (59 isolates), followed by Enterococcus faecium (40 isolates), Enterococcus durans (27 isolates) and Enterococcus hirae (12 isolates). The enterococci strains showed high percentages of resistance to tetracycline (32.6%), to ciprofloxacin (19.6%) and to erythromycin (11.6%). Lower level of resistance (<10%) was detected for ampicillin, chloramphenicol and teicoplanin. One vancomycin-resistant E. faecalis isolate was detected and harbored the vanA resistant gene. Resistance genes detected included tet(M) and/or tet(L), ermB in all tetracycline and erythromycin-resistant isolates. Resistance in E. coli isolates was detected for ampicillin, tetracycline, sulfamethoxazole/trimethoprim, streptomycin, and tobramycin. The blaTEM, aadA, aadA5, strA, strB, tet(A) and/or tet(B), and the intI genes were found in all ampicillin, streptomycin, tetracycline, and sulfamethoxazole/trimethoprim-resistant isolates respectively. The data shown in this study are essential to improve knowledge about the dissemination of resistant strains through wild birds from remote archipelagos such as the Azores Archipelago and the possible implications involved in transferring these resistances either to other animals or to humans.

Use of MALDI-TOF mass spectrometry fingerprinting to characterize Enterococcus spp. and Escherichia coli isolates ☆

UNLABELLED Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a faster and more accurate method to identify intact bacteria than conventional microbiology and/or molecular biology methods. The MALDI-TOF MS method is potentially applicable in diagnostic laboratories to characterize commensal bacterial species, some of which are major pathogens, from human or animal gastrointestinal tracts. The aim of this study was to analyze at the cluster and statistical level the capacity of MALDI-TOF MS to distinguish between previously characterized enterococci and Escherichia coli isolated from wild birds of the Azores archipelago. Soluble proteins were extracted from intact cell cultures of 60 isolates of Enterococcus spp. and 60 isolates of E. coli by an expedient method. MALDI-TOF MS was used to obtain 1200 mass spectra that were statistically analyzed and compared. A total of 215 distinct mass-to-charge (m/z) peaks were obtained, including a peak at m/z 4428±3, which is exclusively found in spectra from Enterococcus isolates, and peaks at m/z 5379±3 and m/z 6253±3, which are only detected in spectra from E. coli isolates. By processing mass spectra and analyzing them statistically with MassUp software, including principal component analysis (PCA) and clustering, it was possible to correctly distinguish between isolates of Enterococcus and Escherichia genera. The results of the proteomic analysis confirm that these tools could be used to characterize whole bacterial cells. In the future, with an optimized protocol for obtaining plasmid-associated proteins and the further development of bioinformatics methods, it is likely that mass peak characteristic of antimicrobial resistance will be detected, increasing the potential usefulness of MALDI-TOF in routine clinical assays. BIOLOGICAL SIGNIFICANCE This study highlights the importance of MALDI-TOF MS in the rapid and reliable identification of bacteria by whole-cell analysis. The mass spectrometry approach performed in this study further contributes for the microbial biomarker discovery culminating in a preferable bacteria identification and antimicrobial resistance tool for the future of clinical microbiology. This article is part of a Special Issue entitled: HUPO 2014.

Comparative proteomics of an extended spectrum β-lactamase producing Escherichia coli strain from the Iberian wolf

UNLABELLED The Iberian wolf (Canis lupus signatus) is an endangered species native to the Iberian Peninsula. Due to their predatory and wild nature, these wolves serve as important indicators of environmental contamination by antimicrobial-resistant bacteria. β-Lactam antibiotics like cefotaxime are the most commonly used antibacterial agents. Bacterial resistance to these antibiotics occurs predominantly through enzymatic inactivation by extended-spectrum beta-lactamases. Escherichia coli strain WA57, isolated from Iberian wolf feces, is a cefotaxime-resistant strain that produces extended-spectrum beta-lactamases. In this study, using 2D-GE combined with MS and bioinformatics, we report significant differences in the abundance of 40 protein spots (p<0.01) from the extracellular, periplasmic, cytoplasmic, and membrane sub-proteomes and the whole-cell proteome of WA57 exposed and non-exposed to cefotaxime. A total of 315 protein spots were collected for protein identification. The comparative proteomics presented gives an overview of the complex changes in expression and metabolism that occur when WA57 is stressed with cefotaxime. Abundance of chaperone, porin and export proteins is particularly affected showing that the stress response and transport functions might directly influence the antibiotic resistance of this strain. BIOLOGICAL SIGNIFICANCE This study highlights the importance of proteomics in detecting protein expression changes in bacterial strains exposed to stress such as that caused by cefotaxime. This approach might help us understand which pathways form barriers for antibiotics. This article is part of a Special Issue entitled: Environmental and structural proteomics.

Azorean wild rabbits as reservoirs of antimicrobial resistant Escherichia coli

Antibiotic resistance in bacteria is an increasing problem that is not only constrained to the clinical setting but also to other environments that can lodge antibiotic resistant bacteria and therefore they may serve as reservoirs of genetic determinants of antibiotic resistance. One hundred and thirty-six faecal samples from European wild rabbits (Oryctolagus cuniculus algirus) were collected on São Jorge Island in Azores Archipelago, and analysed for Escherichia coli isolates. Seventy-seven isolates (56.6%) were recovered and studied for antimicrobial resistance, one isolate per positive sample. Thirteen (16.9%), 19 (24.7%), 25 (32.4%) and 20 (26%) isolates were ascribed to A, B1, B2 and D phylogenetic groups, respectively, by specific primer polymerase chain reaction. Different E. coli isolates were found to be resistant to ampicillin (16.9%), tetracycline (1.3%), streptomycin (42.9%), sulfamethoxazole-trimethoprim (1.3%), amikacin (1.3%), tobramycin (2.6%) and nalidixic acid (1.3%). Additionally, the blaTEM, tetA, strA/strB, aadA, sul1, intI, intI2 and qacEΔ+sul1 genes were found in most resistant isolates. This study showed that E. coli from the intestinal tract of wild rabbits from Azores Archipelago are resistant to widely prescribed antibiotics in medicine and they constitute a reservoir of antimicrobial resistant genes, which may play a significant role in the spread of antimicrobial resistance. Therefore, antibiotic resistant E. coli from Azorean wild rabbits may represent an ecological and public health problem.

A decade-long commitment to antimicrobial resistance surveillance in Portugal

Antimicrobial resistance (AMR) is a worldwide problem with serious health and economic repercussions. Since the 1940s, underuse, overuse, and misuse of antibiotics have had a significant environmental downside. Large amounts of antibiotics not fully metabolized after use in human and veterinary medicine, and other applications, are annually released into the environment. The result has been the development and dissemination of antibiotic-resistant bacteria due to many years of selective pressure. Surveillance of AMR provides important information that helps in monitoring and understanding how resistance mechanisms develop and disseminate within different environments. Surveillance data is needed to inform clinical therapy decisions, to guide policy proposals, and to assess the impact of action plans to fight AMR. The Functional Genomics and Proteomics Unit, based at the University of Trás-os-Montes and Alto Douro in Vila Real, Portugal, has recently completed 10 years of research surveying AMR in bacteria, mainly commensal indicator bacteria such as enterococci and Escherichia coli from the microbiota of different animals. Samples from more than 75 different sources have been accessed, from humans to food-producing animals, pets, and wild animals. The typical microbiological workflow involved phenotypic studies followed by molecular approaches. Throughout the decade, 4,017 samples were collected and over 5,000 bacterial isolates obtained. High levels of AMR to several antimicrobial classes have been reported, including to β-lactams, glycopeptides, tetracyclines, aminoglycosides, sulphonamides, and quinolones. Multi-resistant strains, some relevant to human and veterinary medicine like extended-spectrum β-lactamase-producing E. coli and vancomycin-resistant enterococci, have been repeatedly isolated even in non-synanthropic animal species. Of particular relevance are reports of AMR bacteria in wildlife from natural reserves and endangered species. Future work awaits as this threatening yet unsolved problem persists. GRAPHICAL ABSTRACT Summary diagram of the antimicrobial resistance surveillance work developed by the UTAD Functional Genomics and Proteomics Unit.

Antimicrobial-resistant Escherichia coli and Enterococcus spp. isolated from Miranda donkey (Equus asinus): an old problem from a new source with a different approach.

Purpose. The Miranda donkey (Equus asinus) is an endangeredasinine from Miranda do Douro region, located in the north east of Portugal. We studied the antimicrobial resistance and virulence genes in Escherichia coli and Enterococcus spp. isolates from these animals. Methodology. In March 2014, a total of 66 faecal samples were recovered from independent animals. Antibiotic resistance was determined by the disc diffusion method. Carriage of genes coding for antibiotic‐resistant and virulent factors was analysed by PCR. Results. A total of 66 E. coli and 41 enterococcal isolates were detected, with Enterococcus faecium (61 %) and Enterococcus hirae (24 %) being the most prevalent species. For enterococcal isolates, high percentages of resistance rates to tetracycline (68.3 %), quinupristin/dalfopristin (51.2 %) and ciprofloxacin (48.8 %) were observed. The genes erm(A) and/or erm(B), tet(M) and/or tet(L), vat(D) and/or vat(E) and aph(3′)‐IIIa were also found. The most frequent virulence gene detected was gel(E), followed by ace, cpd and hyl. Escherichia coli isolates were highly resistant to streptomycin (78 %), whereas 39 % of them exhibited resistance to aminoglycosides and tetracycline. Genes sul1 and/or sul2 were detected in 66.7 % of trimethoprim/sulfamethoxazole‐resistant isolates. The virulence genes detected were fim(A) (46 %) and cnf1 (27 %). Conclusion. To the best of our knowledge, this is the first report showing antibiotic resistance among Escherichia coli and Enterococcus spp. isolates from the Miranda donkey in Portugal, indicating possible antibiotic‐resistant bacterial reservoirs. However, the detection of these resistances presents a low risk for other animals and human beings in that rural area.

New Synthesis of Gold‐ and Silver‐Based Nano‐Tetracycline Composites

Abstract A new synthetic methodology of water‐soluble gold and silver nanoparticles (AuNPs@TC and AgNPs@TC), using the antibiotic tetracycline (TC) as co‐reducing and stabilizing agent, is reported. Both colloids exhibit high water stability. The average sizes obtained were 25±10 and 15±5 nm, respectively. Both composites were tested against TC‐resistant bacteria, presenting an increasing antibacterial effect in the case of AgNPs@TC. The sensing towards metal ions was also explored. An interesting and reversible affinity of AuNPs@TC towards AlIII cations in an aqueous system was also observed.

Unravelling the nutriproteomics of chickpea (Cicer arietinum) seeds

Abstract. Chickpea (Cicer arietinum L.) is an essential grain for human consumption owing to its high protein content, nutritional quality and energy-efficient production. The aim of this study was to compare the protein extracts of 24 chickpea genotypes by biochemically characterising the storage proteins. The storage protein content was characterised by protein separation with polyacrylamide gel electrophoresis and visualisation of the banding patterns, which revealed considerable genetic variability within and between genotypes in this chickpea collection. High performance liquid chromatography showed that all genotypes had balanced amino acid content and some were rich in seven amino acids. Two chickpea genotypes, Flip97-171C and Elite, representative of the kabuli and desi types, respectively, were chosen for total proteome analysis. Two-dimensional electrophoresis and subsequent mass spectrometry were used to identify 454 protein spots from the Flip97-171C and Elite proteomes. By using Mascot Server software, 37% of the spots were identified as 47 different proteins involved in a large range of metabolic functions. Most proteins from both proteomes were assigned to nutritional storage activity. Chickpea proteome analysis is essential in reaffirming the quality of this grain protein for human nutrition, and will be important in future nutriproteomics and plant-breeding studies.

Cover Picture: New Synthesis of Gold- and Silver-Based Nano-Tetracycline Composites (ChemistryOpen 3/2016)

The Front Cover picture shows new silver (Ag) and gold (Au) nanoparticle‐based tetracycline (TC) nanocomposites (AuNPs@TC and AgNPs@TC). A new synthetic methodology for the preparation of water‐soluble Au and Ag nanoparticles, using the antibiotic TC as co‐reducing and stabilizing agent, is reported. Both colloids exhibit high water stability, with an average size of 15±5 and 25±10 nm, respectively. Both composites are tested against TC‐resistant bacteria, presenting an increasing antibacterial effect in the case of AgNPs@TC. Moreover, the sensing towards metal ions is also explored by using AuNPs@TC, which is selective and reversible for aluminum(III) in water. More information can be found in the Full Paper by C. Lodeiro, J. Fernández‐Lodeiro, and co‐workers on page 206 in Issue 3, 2016 (DOI: 10.1002/open.201600016).