How different is the proteome of the extended spectrum ß-lactamase producing Escherichia coli strains from seagulls of the Berlengas natural reserve of Portugal?

UNLABELLED: ß-Lactam antibiotics like cefotaxime are the most commonly used antibacterial agents. Escherichia coli strains 5A, 10A, 12A and 23B isolated from Seagulls feces, are cefotaxime-resistant strains that produces extended-spectrum beta-lactamases. Bacterial resistance to these antibiotics occurs predominantly through structural modification on the penicillin-binding proteins and enzymatic inactivation by extended-spectrum ß-lactamases. Using classical proteomic techniques (2D-GE) coupled to mass spectrometry and bioinformatics extended analysis, in this study, we report several significant differences in cytoplasmic proteins expression when the strains were submitted to antibiotic stress and when the resistant strains were compared with a non-resistant strain. A total of 79 differentially expressed spots were collected for protein identification. Significant level of expression was found in antibiotic resistant proteins like ß-lactamase CTX-M-1 and TEM and also in proteins related with oxidative stress. This approach might help us understand which pathways form barriers for antibiotics, another possible new pathways involved in antibiotic resistance to devise appropriate strategies for their control already recognized by the World Health Organization and the European Commission. BIOLOGICAL SIGNIFICANCE: This study highlights the protein differences when a resistant strain is under antibiotic pressure and how different can be a sensible and resistant strain at the protein level. This survey might help us to understand the specifics barriers for antibiotics and which pathways are involved in its resistance crosswise the wildlife.

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

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. ß-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.

A comprehensive factorial design study of variables affecting protein extraction from formalin-fixed kidney tissue samples.

Formalin-fixed tissues are an important source of biological samples for biomedical research. However, proteomics analysis of formalin-fixed tissues has been set aside by formalin-induced protein modifications, which reduce protein extraction efficiency. In this study, a two level full factorial experimental design (2(4)) was used to determine the effects of the extracting conditions in the efficiency of protein recovery from formalin-fixed kidney samples. The following variables were assessed: temperature of extraction, pH of extraction, composition of the extracting buffer and the use ultrasonic energy applied with probe. It is clearly demonstrated that when hating and ultrasonic energy are used in conjunction, a 7-fold increase (p < 0.05) in protein extraction is obtained if compared to extracting conditions for which neither heating nor ultrasonic energy are used. The optimization study was done following the amount of protein extracted by UV (Nanodrop(®) technology, protein ABS at 280 nm) and by 1D SDS-PAGE. Extracts obtained with the optimized conditions were subjected to LC-MALDI MS/MS. A total of 112 proteins were identified.

Mass spectrometry-based fingerprinting of proteins & peptides in wine quality control: a critical overview.

In this work we have critically revised and updated the literature dealing with wine quality control based on protein or peptide mass spectrometry-based fingerprinting. A number of pitfalls in the experimental design of most work dealing with this subject are highlighted along with recommendations on how to circumvent them. As a general trend, the conclusions reported to date in the literature of the topic are inconclusive mainly due to the (i) low number of representative samples, (ii) lack of basic analytical concepts, and (iii) lack of adequate statistical and software tools. In addition, we have critically revised the sample treatments commonly used to separate proteins from wines, emphasizing that the majority of literature is devoted to white wines, probably because of difficulties in isolating the protein content in red wines.

Antimicrobial resistance in faecal enterococci and Escherichia coli isolates recovered from Iberian wolf.

The aim of this study was to report the antimicrobial resistance, the molecular mechanisms associated and the detection of virulence determinants within faecal Enterococcus spp. and Escherichia coli isolates of Iberian wolf. Enterococci (n = 227) and E. coli (n = 195) isolates were obtained from faecal samples of Iberian wolf (Canis lupus signatus). High rates of resistance were detected for tetracycline and erythromycin among the enterococci isolates, and most of resistant isolates harboured the tet(M) and/or tet(L) and erm(B) genes, respectively. The blaTEM, tet(A) and/or tet(B), and aadA or strA-strB genes were detected among most ampicillin-, tetracycline- or streptomycin-resistant E. coli isolates, respectively. E. coli isolates were ascribed to phylogroups A (n = 56), B1 (91), B2 (13) and D (35). The occurrence of resistant enterococci and E. coli isolates in the faecal flora of Iberian wolf, including the presence of resistant genes in integrons, and virulence determinants was showed in this study. Iberian wolf might act as reservoir of certain resistance genes that could be spread throughout the environment.

Direct matrix assisted laser desorption ionization mass spectrometry-based analysis of wine as a powerful tool for classification purposes.

The variables affecting the direct matrix assisted laser desorption ionization mass spectrometry-based analysis of wine for classification purposes have been studied. The type of matrix, the number of bottles of wine, the number of technical replicates and the number of spots used for the sample analysis have been carefully assessed to obtain the best classification possible. Ten different algorithms have been assessed as classification tools using the experimental data collected after the analysis of fourteen types of wine. The best matrix was found to be α-Cyano with a sample to matrix ratio of 1:0.75. To correctly classify the wines, profiling a minimum of five bottles per type of wine is suggested, with a minimum of three MALDI spot replicates for each bottle. The best algorithm to classify the wines was found to be Bayes Net.

Proteome of a methicillin-resistant Staphylococcus aureus clinical strain of sequence type ST398.

Proteomics is a powerful tool to analyze the differences in gene expression of bacterial strains. Staphylococcus aureus has long been recognized as an important pathogen in human disease. In order to investigate this pathogen, the proteome of a clinical methicillin-resistant S. aureus (MRSA) strain of the sequence type ST398 was determined using 2-DE. Using 2-DE we obtained a total of 105 spots the MRSA strain. Furthermore in correlation with bioinformatic databases, they allowed accurate identification and characterization of proteins, resulting in 227 identified proteins. There were found proteins related to basic function of the cell, but also proteins related to virulence like catalase, specific of S. aureus species, and proteins related to antibiotic resistance. Proteins associated with antibiotic resistance or virulence factors are related to genomic databases. The most abundant classes identified involved glycolysis, energy production, one-carbon metabolism, and oxidation-reduction process, all of which reflect an active metabolism. These results highlight the importance of proteomics to deepen in the knowledge of protein expression of MRSA strain of the lineage ST398, microorganism with diverse and important resistance mechanisms. With this proteome map we have an essential tool for a better understanding of this pathogen and providing new data for protein databases. This article is part of a Special Issue entitled: Proteomics: The clinical link.

Detection of extended-spectrum beta-lactamase-producing Escherichia coli isolates in faecal samples of Iberian lynx.

AIMS: To characterize the diversity of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli isolates recovered within the faecal microbiota of Iberian lynx. The identification of other associated resistance genes and the analysis of clonal relationship were also focused in this study. METHODS AND RESULTS: From 2008 to 2010, 128 faecal samples of Iberian lynx (wild and captive animals) were collected. Eleven tested samples contained cefotaxime-resistant E. coli isolates (all belonging to captive animals) and 10 ESBL-producing isolates were showed. CTX-M-14 and SHV-12 ESBL-types were detected and seven different patterns were identified by pulsed-field gel electrophoresis analysis. CONCLUSIONS: The occurrence of unrelated multiresistant E. coli in faecal flora of captive specimens of Iberian lynx, including the presence of ESBLs, resistant genes in integrons and virulence determinants was showed in this study. SIGNIFICANCE AND IMPACT OF THE STUDY: The results obtained in this study highlight the environmental problem as future reintroductions of Iberian lynx could lead to a spread of resistant bacteria. Additionally, ESBL-producing bacteria can represent a health problem for this endangered species.

Genetic characterisation of extended-spectrum ß-lactamases in Escherichia coli isolated from retail chicken products including CTX-M-9 containing isolates: a food safety risk factor.

1. Bacterial resistance to ß-lactam antibiotics has risen dramatically in Escherichia coli from food animals. In a previous study, 29 randomly selected chicken products, collected in Portugal, were analysed for the presence of extended-spectrum ß-lactamases (ESBLs)-producing E. coli; and during this study the genetic characterisation of ESBLs genes was investigated. 2. The presence of genes encoding TEM, OXA, SHV, and CTX-M type beta-lactamases was studied by PCR followed by sequencing. Additionally, other mechanisms of antimicrobial resistance, phylogenetic groups and the presence of virulence determinants were evaluated among the isolates. 3. ß-lactamases genes were identified as follows: bla (CTX-M-14) (n = 4), bla (CTX-M-1) (n = 2), bla (CTX-M-9) (n = 4) and bla (TEM-52) (n = 13). Mutations at positions -42, -18, -1, and +58 of ampC promoter region were identified in 4 non-ESBL-producing isolates. The tet(A) or tet(B) genes were identified in all tetracycline-resistant isolates; the aadA gene detected in 8 of 10 streptomycin-resistant isolates; the aac(3)-II gene in all gentamicin-resistant isolates; the cmlA gene in the chloramphenicol-resistant isolate; and sul1 and/or sul2 and/or sul3 genes were found in all trimethoprim-sulfamethoxazole-resistant isolates. The intI1 gene was detected in 8 trimethoprim-sulfamethoxazole-resistant isolates and the intI2 gene in 4 isolates; one gene cassette arrangements were identified among class 1 integrons (dfrA1 + aadA1) and among the class 2 integrons (dfrA1 + sat2 + aadA1). Among cefotaxime-resistant isolates, 16 belonged to A or B1 phylogenetic groups, while 11 isolates were classified into the D or B2 phylogroups. At least one virulence-associated gene (aer, fimA, or papC) was detected in 74·1% of the cefotaxime-resistant isolates. 4. Because ESBLs-producing bacteria are resistant to a broad range of ß-lactams, infections caused by these organisms complicate therapy and limit treatment options.

Antimicrobial activity and occurrence of bacteriocin structural genes in Enterococcus spp. of human and animal origin isolated in Portugal.

The main objective of this study was to detect the antimicrobial activity and the presence of bacteriocin structural genes in 224 enterococcal isolates from fecal origin obtained from humans, pets, wild animals and birds. Direct antimicrobial activity against Listeria monocytogenes CECT4032 was detected in 102 (45.6%) of the tested isolates. From these, only 22 displayed bacteriocin activity against this indicator. The bacteriocinogenic strains contained one or more of the bacteriocin structural genes tested in this study, with those of enterocins P, A and L50 (L50A and L50B) being the most abundant. Our results show a high occurrence of the combination of different bacteriocin structural genes in the enterococcal isolates analyzed, indicating an elevated genetic potential of these strains to produce various bacteriocins.

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.

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. METHODS AND RESULTS: The presence of VRE was investigated in 33 faecal samples of B. buteo. Samples were seeded in Slanetz-Bartley agar plates supplemented with vancomycin for VRE recovery. Genes encoding antimicrobial resistance and virulence were studied by polymerase chain reaction. Vancomycin-resistant Enterococcus faecium isolates were characterized by multilocus sequence typing. VRE with an acquired mechanism of resistance (vanA genotype) were detected in 9% of samples analysed (Ent. faecium and Enterococcus durans). In addition, 27% of samples contained VRE with an intrinsic mechanism of resistance (Enterococcus gallinarum, vanC1). All vanA-containing isolates showed resistance to tetracycline and erythromycin and harboured the tet(M) and/or tet(L) genes, in addition to the ermB gene. The vat(E) and/or vat(D), cat(A) and aph(3')-IIIa genes were identified in quinupristin-dalfopristin-, chloramphenicol-, and kanamycin-resistant vanA-containing strains, respectively. The sequence types ST273 and ST5 were identified in two vanA-positive Ent. faecium isolates, and the presence of hyl, gelE, cylA, cylL and cylM virulence genes and gelatinase activity were identified in Ent. faecium ST5 strain. CONCLUSIONS: The intestinal tract of B. buteo could be a reservoir of vanA-positive enterococci. SIGNIFICANCE AND IMPACT OF THE STUDY: First study focused to define the occurrence of vanA-containing Enterococcus strains in B. buteo.

Detection and genetic characterisation of vanA-containing Enterococcus strains in healthy Lusitano horses.

Lusitano horses were investigated in order to detect the presence of vancomycin-resistant enterococci. vanA isolates showed high level vancomycin (Minimum inhibitory concentration; MIC > or = 128 mg/l) and teicoplanin resistance (MIC 64 mg/l), as well as resistance to ciprofloxacin, erythromycin and tetracycline. The tet(L) and erm(B) genes, associated with tetracycline and erythromycin resistance, respectively, were found in all vanA isolates. The intestinal tract of Lusitano horses can be a potential reservoir for vanA-containing enterococci.

Genetic characterization of antibiotic resistance in enteropathogenic Escherichia coli carrying extended-spectrum beta-lactamases recovered from diarrhoeic rabbits.

A total of 52 Escherichia coli strains isolated from diarrhoeic rabbits were investigated for their enteropathogenic E. coli (EPEC) pathotype by PCR amplification of eae and bfp virulence genes. A total of 22 EPEC isolates were identified, serotyped and studied for antibiotic resistance and screened for the detection of extended-spectrum beta-lactamases (ESBLs). The EPEC isolates belonged to three serogroups (O26, O92 and O103). The most common serogroup (O103:K-:H2) was observed among 17 EPEC strains, the O92:K-serogroup in three isolates (the antibiotic sensitive ones) and the remaining O26:K-serogroup in two isolates (the ESBLs isolates). Resistances to ampicillin and tetracycline were the most frequent and detected followed by resistance to nalidixic acid, streptomycin, trimethoprim-sulphamethoxazole, cefoxitin, gentamicin and ciprofloxacin. All the isolates were sensitive for amikacin, ceftazidime, aztreonam, imipenem, chloramphenicol, tobramycin and amoxicillin + clavulanic acid. Two isolates recovered from two adult animals showed an intermediate susceptibility to cefotaxime, and a positive screening test for ESBL was demonstrated in both. The bla(TEM) gene was demonstrated in the majority of ampicillin-resistant isolates. The aac(3)-II or aac(3)-IV genes were detected in the four gentamicin-resistant isolates. In addition, the aadA gene was detected in 60% of streptomycin-resistant isolates. The tet(A) or tet(B) genes were identified in all tetracycline-resistant isolates. A total of nine EPEC isolates showed the phenotype SXT-resistant, and the sul1 and/or sul2 and/or sul3 genes were detected in all of them. Our findings showed that the molecular detection by the eae and bfp genes by PCR followed by serotyping is useful for monitoring trends in EPEC infections of rabbits allowing the identification of their possible reservoirs. The detection of genes involved in the resistance to antibiotics of different families in a relatively high proportion of faecal E. coli isolates of rabbits is of great interest and could be considered a serious public health problem.

Antimicrobial resistance and phylogenetic groups in isolates of Escherichia coli from seagulls at the Berlengas nature reserve.

Fifty-three faecal samples from yellow-legged gulls (Larus cachinnans) at the Berlengas nature reserve in Portugal were cultured on Levine agar plates not supplemented with antimicrobial agents, and one Escherichia coli colony was isolated and identified from each sample. The percentages of resistant isolates for each of the drugs were ampicillin (43.4 per cent), tetracycline (39.6 per cent), nalidixic acid (34.0 per cent), streptomycin (32.1 per cent), trimethoprim-sulfamethoxazole (SXT) (26.4 per cent), ciprofloxacin (18.9 per cent), chloramphenicol (18.9 per cent), gentamicin (7.5 per cent), tobramycin (7.5 per cent) amikacin (5.7 per cent) and amoxicillin-clavulanic acid (1.9 per cent). All the isolates were susceptible to cefoxitin, ceftazidime, cefotaxime, aztreonam and imipenem. The following resistance genes were detected: bla(TEM) (17 of 23 ampicillin-resistant isolates), tet(A) and/or tet(B) (18 of 21 tetracycline-resistant isolates), aadA (12 of 17 streptomycin-resistant isolates), cmlA (all chloramphenicol-resistant isolates), aac(3)-II with or without aac(3)-IV (all four gentamicin-resistant isolates), and sul1 and/or sul2 and/or sul3 (all 14 SXT-resistant isolates). The intI1 gene was detected in 10 of 14 SXT-resistant isolates, and three of them also contained class 2 integrons; four different gene cassette arrangements were identified among class 1 integrons (aadA, dfrA1+aadA1, dfrA12+orfF+aadA2 and sat+psp+aadA2) and one among the class 2 integrons (dfrA1+sat+aadA1). Ninety per cent of the isolates were included in the A or B1 phylogenetic groups.

High levels of genetic diversity throughout the range of the Portuguese wheat landrace 'Barbela'.

BACKGROUND AND AIMS: Landrace populations represent an important intra-crop reservoir of biodiversity and source of novel gene alleles for use in breeding programmes. Here the aim was to measure the diversity of a wheat landrace, 'Barbela', from the north of Portugal. METHODS: DNA was extracted from 59 accessions of Barbela collected across its geographical range. Diversity was measured by microsatellite length polymorphisms using 27 primer pairs amplifying 34 polymorphic microsatellite loci. KEY RESULTS: High levels of polymorphism were found, with an average polymorphism information content of 0.52; an average of 4.77 alleles (range 2-11) were present at each locus, and half of these loci showed an additional allele in the reference variety 'Chinese Spring'. CONCLUSIONS: 'Barbela' is maintained from seeds collected by farmers, but it maintains high allelic variation, and no groupings of accessions were detected when analysed by geographical region, farm or climate, indicating that the wheat landrace is a homogeneous entity. The diversity within the farmer-maintained landrace demonstrates the importance of characterization and maintenance of landrace collections before valuable genetic combinations are lost as uniform commercial crops are introduced.

Mapping QTLs for grain hardness and puroindoline content in wheat ( Triticum aestivum L.).

Genes for puroindoline-a (Pin-a), puroindoline-b (Pin-b) and grain-softness proteins (GSP) have been shown to be linked to the dominant Ha locus responsible for the soft texture of the grain. Though linkage has been demonstrated of the puroindoline genes to the Ha locus, there is no clear evidence that puroindoline content is the product of the gene Ha. A segregating population of 115 recombinant inbred lines (RILs) originating from a cross between the hexaploid Synthetic wheat ( Triticum durum x Aegilops tauschii, W 7984) and the cultivar 'Opata' (M 85) was studied in two different experimental years to detect Quantitative Trait Loci (QTLs) for three traits: grain hardness (Hard), puroindoline-a (Pin-a) and puroindoline-b (Pin-b) contents. The detection of QTLs was performed using marker linear regression. Negative correlation coefficients (-0.86 and -0.80) were identified between grain hardness and puroindoline content (a and b, respectively) on data obtained in 1996. Results obtained in 1999 confirmed the negative correlation between Hard and Pin-a (-0.73); however a positive correlation coefficient was found with Pin-b content (0.41). Total phenotypic variation explained by each QTL was calculated (R2). For each of the Hard, Pin-a and Pin-b traits one major QTL was detected on the short arm of chromosome 5D, located close to the mta9 allele (puroindoline-a). For the first year (1996) the QTL in this region explained around 63% of the phenotypic variability in grain hardness, 77% in Pin-a and 45% in Pin-b contents. These values were confirmed in trials carried out in 1999 with a R2 value of 0.71, 0.72 and 0.25 for Hard, Pin-a and Pin-b, respectively. In 1996 and 1999 a second major QTL was detected for grain hardness on the long arm of the same chromosome. Present results indicate that it cannot be definitely concluded that puroindoline content represents a linear explanation for variations in grain hardness.