Luís Pinto

Wild boars as reservoirs of extended‐spectrum beta‐lactamase (ESBL) producing Escherichia coli of different phylogenetic groups

ESBL‐producing E. coli isolates have been isolated from eight of seventy seven faecal samples (10.4%) of wild boars in Portugal. The ESBL types identified by PCR and sequencing were blaCTX‐M‐1 (6 isolates) and blaCTX‐M‐1 + blaTEM1‐b (2 isolates). Further resistance genes detected included tet (A) or tet (B) (in three tetracycline‐resistant isolates), aad A (in three streptomycin‐resistant isolates), cml A (in one chloramphenicol‐resistant isolate), sul 1 and/or sul 2 and/or sul 3 (in all sulfonamide‐resistant isolates). The intI 1 gene encoding class 1 integrase was detected in all ESBL‐producing E. coli isolates. One isolate also carried the intI 2 gene, encoding class 2 integrase. The ESBL‐producing E. coli isolates could be assigned to phylogenetic groups B1 (3 isolates), B2 (3 isolates) or A (2 isolates). Amino acid change in GyrA protein (Ser83Leu or Asp87Tyr) was detected in three nalidixic acid‐resistant and ciprofloxacin‐susceptible isolates. Two amino acid changes in GyrA (Ser83Leu + Asp87Asn) and one in ParC (Ser80Ile) were identified in two nalidixic acid‐ and ciprofloxacin‐resistant isolates. As evidenced by this study wild boars could be a reservoir of antimicrobial resistance genes. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Genetic Detection of Extended-Spectrum β-Lactamase-Containing Escherichia coli Isolates from Birds of Prey from Serra da Estrela Natural Reserve in Portugal

ABSTRACT Extended-spectrum β-lactamase-containing Escherichia coli isolates were detected in 32 of 119 fecal samples (26.9%) from birds of prey at Serra da Estrela, and these isolates contained the following β-lactamases: CTX-M-1 (n = 13), CTX-M-1 plus TEM-1 (n = 14), CTX-M-1 plus TEM-20 (n = 1), SHV-5 (n = 1), SHV-5 plus TEM-1 (n = 2), and TEM-20 (n = 1).

Proteomic characterization of vanA-containing Enterococcus recovered from Seagulls at the Berlengas Natural Reserve, W Portugal

BackgroundEnterococci have emerged as the third most common cause of nosocomial infections, requiring bactericidal antimicrobial therapy. Although vancomycin resistance is a major problem in clinics and has emerged in an important extend in farm animals, few studies have examined it in wild animals. To determine the prevalence of van A-containing Enterococcus strains among faecal samples of Seagulls (Larus cachinnans) of Berlengas Natural Reserve of Portugal, we developed a proteomic approach integrated with genomic data. The purpose was to detect the maximum number of proteins that vary in different enterococci species which are thought to be connected in some, as yet unknown, way to antibiotic resistance.ResultsFrom the 57 seagull samples, 54 faecal samples showed the presence of Enterococcus isolates (94.7%). For the enterococci, E. faecium was the most prevalent species in seagulls (50%), followed by E. faecalis and E. durans (10.4%), and E. hirae (6.3%). VanA-containing enterococcal strains were detected in 10.5% of the 57 seagull faecal samples studied. Four of the vanA-containing enterococci were identified as E. faecium and two as E. durans. The tet(M) gene was found in all five tetracycline-resistant vanA strains. The erm(B) gene was demonstrated in all six erythromycin-resistant vanA strains. The hyl virulence gene was detected in all four van A-containing E. faecium isolates in this study, and two of them harboured the pur K1 allele. In addition these strains also showed ampicillin and ciprofoxacin resistance. The whole-cell proteomic profile of van A-containing Enterococcus strains was applied to evaluate the discriminatory power of this technique for their identification. The major differences among species-specific profiles were found in the positions corresponding to 97-45 kDa. Sixty individualized protein spots for each vanA isolate was identified and suitable for peptide mass fingerprinting measures by spectrometry measuring (MALDI/TOF MS) and their identification through bioinformatic databases query. The proteins were classified in different groups according to their biological function: protein biosynthesis, ATP synthesis, glycolysis, conjugation and antibiotic resistance. Taking into account the origin of these strains and its relation to infectious processes in humans and animals, it is important to explore the proteome of new strains which might serve as protein biomarkers for biological activity.ConclusionsThe comprehensive description of proteins isolated from vancomycin-resistant Enterococcus faecium and E. durans may provide new targets for development of antimicrobial agents. This knowledge may help to identify new biomarkers of antibiotic resistance and virulence factors.

MLST and a genetic study of antibiotic resistance and virulence factors in vanA-containing Enterococcus from buzzards (Buteo buteo)

Aims:  To analyse the occurrence of faecal carriage of vancomycin‐resistant enterococci (VRE) in Buteo buteo and to study the associated resistance and virulence genes.

Genomic and proteomic evaluation of antibiotic resistance in Salmonella strains

Using Salmonella strains identical to those present in the gastrointestinal tract of different animals we aim to determine and compare the proteome of two serotypes, Salmonella Typhimurium and Enteritidis recovered from faecal samples of wild boars and wild rabbits, respectively. The presence of genes responsible for antibiotic resistance was detected by PCR. Proteomes of the two distinct serotypes were determined using 2-DE in order to identify proteins associated with antibiotic resistance or virulence. Through 2-DE we obtained a total of 229 spots from both strains. All were suitable for MALDI-TOF/TOF and, in correlation with bioinformatic databases, allowed accurate identification and characterization of proteins. S. Enteritidis recovered from wild rabbits was sensitive to all the antibiotics tested in contrast to S. Typhimurium isolated from wild boars which presented a resistance phenotype to ampicillin, streptomycin and chloramphenicol. Nevertheless, despite the different ratio of proteins observed in each proteome according to their biological function, no significant difference was observed in the involvement of these proteins in pathogenicity. Bearing in mind that serotypes are related to infectious processes in humans and animals, it is important to explore the proteome of new strains which might serve as protein biomarkers for biological activity.

Clonal Lineages, Antibiotic Resistance and Virulence Factors in Vancomycin-Resistant Enterococci Isolated from Fecal Samples of Red Foxes (Vulpes Vulpes)

Fourteen vanA-containing entero-coccal isolates were detected in seven of 52 fecal samples (13.5%) from free-ranging red foxes in Portugal. Nine of the vanA-containing isolates were Enterococcus faecium and five were E. durans. Both sequence types, ST262 and ST273, were identified among E. faecium isolates.

After genomics, what proteomics tools could help us understand the antimicrobial resistance of Escherichia coli?☆

Proteomic approaches have been considerably improved during the past decade and have been used to investigate the differences in protein expression profiles of cells grown under a broad spectrum of growth conditions and with different stress factors including antibiotics. In Europe, the most significant disease threat remains the presence of microorganisms that have become resistant to antimicrobials and so it is important that different scientific tools are combined to achieve the largest amount of knowledge in this area of expertise. The emergence and spread of the antibiotic-resistant Gram-negative pathogens, such as Escherichia coli, can lead to serious problem public health in humans. E. coli, a very well described prokaryote, has served as a model organism for several biological and biotechnological studies increasingly so since the completion of the E. coli genome-sequencing project. The purpose of this review is to present an overview of the different proteomic approaches to antimicrobial-resistant E. coli that will be helpful to obtain a better knowledge of the antibiotic-resistant mechanism(s). This can also aid to understand the molecular determinants involved with pathogenesis, which is essential for the development of effective strategies to combat infection and to reveal new therapeutic targets. This article is part of a Special Issue entitled: Proteomics: The clinical link.

Potential spoilage yeasts in winery environments: Characterization and proteomic analysis of Trigonopsis cantarellii

Wine microbiota is complex and includes a wide diversity of yeast species. Few of them are able to survive under the restrictive conditions of dry red wines. In our study we detected and identified seven yeast species of the order Saccharomycetales that can be considered potential spoilers of wines due to physiological traits such as acidogenic metabolism and off-odor generation: Arthroascus schoenii, Candida ishiwadae, Meyerozyma guilliermondii, Pichia holstii, Pichia manshurica, Trigonopsis cantarellii, and Trigonopsis variabilis. Based on the prevalence of T. cantarellii isolates in the wine samples of our study, we further characterized this species, determined molecular and phenotypic features, and performed a proteomic analysis to identify differentially expressed proteins at mid-exponential growth phase in the presence of ethanol in the culture broth. This yeast species is shown to be able to grow in the presence of ethanol by expressing heat shock proteins (Hsp70, Hsp71) and a DNA damage-related protein (Rad24), and to be able to confer spoilage characteristics on wine.

The Role of Proteomics in Elucidating Multiple Antibiotic Resistance in Salmonella and in Novel Antibacterial Discovery

Rui Pacheco1-4**, Susana Correia1-4**, Patricia Poeta3,4, Luis Pinto1-4 and Gilberto Igrejas1,2, 1Institute for Biotechnology and Bioengineering, Center of Genomics and Biotechnology University of Tras-os-Montes and Alto Douro 2Department of Genetics and Biotechnology, University of Tras-os-Montes and Alto Douro 3Center of Studies of Animal and Veterinary Sciences 4Veterinary Science Department, University of Tras-os-Montes and Alto Douro Portugal

Effect of allelic variation at glutenin and puroindoline loci on bread-making quality: favorable combinations occur in less toxic varieties of wheat for celiac patients

Genetically diverse wheat samples, twenty-seven Triticum aestivum L. varieties, grown in two environments (Portugal and Spain) were analyzed for their allelic composition in high-molecular-weight glutenin subunits (HMW-GS), low-molecular-weight glutenin subunits (LMW-GS) and puroindolines, as well as their protein content, hardness, sodium dodecyl sulfate-sedimentation (SDS-S), mixograph mixing time and breakdown resistance (MT and BDR, respectively) parameters, and R5 reactivity. The environment showed significant effect on protein content, SDS-S and BDR parameters. In relation to HMW-GS quality effect, the allelic composition Glu-A1d, Glu-B1al, Glu-D1d presented the best results. From the complex Glu-3 loci (LMW-GS), only Glu-B3 locus showed a significant effect on the quality parameters. The Glu-B3ab allele is desirable considering the higher mean values for SDS-S, MT and hardness, and the lower mean values for BDR. Regarding puroindolines, Pina-D1a, Pinb-D1c allelic composition (Leu to Pro at position +60) showed the best quality potential. In addition, we found negative (SDS-S and MT) and positive (BDR) significant correlations between quality parameters and the amount of potential celiac disease toxic epitopes, suggesting that wheat breeding aiming at quality does not have a negative impact on wheat toxicity and on the other hand, emphasizes the need for a more comprehensive wheat breeding programs that encompass celiac disease problematic.