Prediction of Staphylococcus aureus antimicrobial resistance by whole-genome sequencing

Whole-genome sequencing (WGS) could potentially provide a single platform for extracting all the information required to predict an organism’s phenotype. However, its ability to provide accurate predictions has not yet been demonstrated in large independent studies of specific organisms. In this study, we aimed to develop a genotypic prediction method for antimicrobial susceptibilities. The whole genomes of 501 unrelated Staphylococcus aureus isolates were sequenced, and the assembled genomes were interrogated using BLASTn for a panel of known resistance determinants (chromosomal mutations and genes carried on plasmids). Results were compared with phenotypic susceptibility testing for 12 commonly used antimicrobial agents (penicillin, methicillin, erythromycin, clindamycin, tetracycline, ciprofloxacin, vancomycin, trimethoprim, gentamicin, fusidic acid, rifampin, and mupirocin) performed by the routine clinical laboratory. We investigated discrepancies by repeat susceptibility testing and manual inspection of the sequences and used this information to optimize the resistance determinant panel and BLASTn algorithm. We then tested performance of the optimized tool in an independent validation set of 491 unrelated isolates, with phenotypic results obtained in duplicate by automated broth dilution (BD Phoenix) and disc diffusion. In the validation set, the overall sensitivity and specificity of the genomic prediction method were 0.97 (95% confidence interval [95% CI], 0.95 to 0.98) and 0.99 (95% CI, 0.99 to 1), respectively, compared to standard susceptibility testing methods. The very major error rate was 0.5%, and the major error rate was 0.7%. WGS was as sensitive and specific as routine antimicrobial susceptibility testing methods. WGS is a promising alternative to culture methods for resistance prediction in S. aureus and ultimately other major bacterial pathogens.

Resistance to the anticoagulant rodenticides – the deployment of the new molecular methodology to identify mutations of the VKORC1 ‘resistance gene’, and understanding their potential impact on treatment outcome

Anticoagulants rodenticides have already known for over half a century, as effective and safe method of rodent control. However, discovered in 1958 anticoagulant resistance has given us a very important problem for their future long-term use. Laboratory tests provide the main method for identification the different types of anticoagulant resistances, quantify the magnitude of their effect and help us to choose the best pest control strategy. The main important tests are lethal feeding period (LFP) and blood clotting response (BCR) tests. These tests can now be used to quantify the likely effect of the resistance on treatment outcome by providing an estimate of the ‘resistance factor’. In 2004 the gene responsible for anticoagulant resistance (VKORC1) was identified and sequenced. As a result, a new molecular resistance testing methodology has been developed, and a number of resistance mutations, particularly in Norway rats and house mice. Three mutations of the VKORC1 gene in Norway rats have been identified to date that confer a degree of resistance to bromadiolone and difenacoum, sufficient to affect treatment outcome in the field.

Aberrant brain responses to emotionally valent words is normalised after cognitive behavioural therapy in female depressed adolescents

AbstractBackground Depression in adolescence is debilitating with high recurrence in adulthood, yet its pathophysiological mechanism remains enigmatic. To examine the interaction between emotion, cognition and treatment, functional brain responses to sad and happy distractors in an affective go/no-go task were explored before and after Cognitive Behavioural Therapy (CBT) in depressed female adolescents, and healthy participants. Methods Eighty-two Depressed and 24 healthy female adolescents, aged 12 to 17 years, performed a functional magnetic resonance imaging (fMRI) affective go/no-go task at baseline. Participants were instructed to withhold their responses upon seeing happy or sad words. Among these participants, 13 patients had CBT over approximately 30 weeks. These participants and 20 matched controls then repeated the task. Results At baseline, increased activation in response to happy relative to neutral distractors was observed in the orbitofrontal cortex in depressed patients which was normalized after CBT. No significant group differences were found behaviourally or in brain activation in response to sad distractors. Improvements in symptoms (mean: 9.31, 95% CI: 5.35-13.27) were related at trend-level to activation changes in orbitofrontal cortex. Limitations In the follow-up section, a limited number of post-CBT patients were recruited. Conclusions To our knowledge, this is the first fMRI study addressing the effect of CBT in adolescent depression. Although a bias toward negative information is widely accepted as a hallmark of depression, aberrant brain hyperactivity to positive distractors was found and normalised after CBT. Research, assessment and treatment focused on positive stimuli could be a future consideration. Moreover, a pathophysiological mechanism distinct from adult depression may be suggested and awaits further exploration.

The commensal infant gut meta-mobilome as a potential reservoir for persistent multidrug resistance integrons

Despite the accumulating knowledge on the development and establishment of the gut microbiota, its role as a reservoir for multidrug resistance is not well understood. This study investigated the prevalence and persistence patterns of an integrase gene (int1), used as a proxy for integrons (which often carry multiple antimicrobial resistance genes), in the fecal microbiota of 147 mothers and their children sampled longitudinally from birth to 2 years. The study showed the int1 gene was detected in 15% of the study population, and apparently more persistent than the microbial community structure itself. We found int1 to be persistent throughout the first two years of life, as well as between mothers and their 2-year-old children. Metagenome sequencing revealed integrons in the gut meta-mobilome that were associated with plasmids and multidrug resistance. In conclusion, the persistent nature of integrons in the infant gut microbiota makes it a potential reservoir of mobile multidrug resistance.

The Spanish Guerrilla 1808-1814 as a model for people’s wars and resistance movements

Transforming the meaning of the term 'guerrilla' which had once meant feud or private warfare, and then irregular war conducted by special forces on behalf of a state or government, the Spanish Guerrilla (part of the Peninsular War) against Napoleon became the model to be emulated by insurgency movements across the world. Even though the term itself continued to be used, even in Spanish, for special operations, in henceforth became imbued with an ideological dimension, which is how it would be used especially in the 20th century.

Rapid, automated detection of stem canker symptoms in woody perennials using artificial neural network analysis

Background Pseudomonas syringae can cause stem necrosis and canker in a wide range of woody species including cherry, plum, peach, horse chestnut and ash. The detection and quantification of lesion progression over time in woody tissues is a key trait for breeders to select upon for resistance. Results In this study a general, rapid and reliable approach to lesion quantification using image recognition and an artificial neural network model was developed. This was applied to screen both the virulence of a range of P. syringae pathovars and the resistance of a set of cherry and plum accessions to bacterial canker. The method developed was more objective than scoring by eye and allowed the detection of putatively resistant plant material for further study. Conclusions Automated image analysis will facilitate rapid screening of material for resistance to bacterial and other phytopathogens, allowing more efficient selection and quantification of resistance responses.

Differentiation in populations of the apple scab fungus Venturia inaequalis on cultivars in a mixed orchard remains over time

The ascomycete Venturia inaequalis causes annual epidemics of apple scab worldwide. Scab development is reduced in mixed cultivar orchards compared with monocultures. To use mixtures in commercial production, we need to understand how the population of scab changes in a mixed orchard and how likely a super race, with virulence factors overcoming multiple resistance factors in the mixed orchard, is to emerge and become dominant. We used short sequence repeat (SSR) markers to investigate the temporal change of scab populations in two mixed cultivar orchards in the UK to infer the likelihood of emergence of a scab super race. There were no significant differences between the populations at the two sampling times (six or seven years apart) in either of the two mixed orchards. In one of the orchards apple scab populations on different cultivars were significantly different and the differences did not diminish over time. These results suggest that it is not inevitable that a super race of V. inaequalis will become dominant during the lifetime of a commercial apple orchard.

Detection of SDHI insensitivity in a Zymoseptoria tritici field population associated with the SdhC-H152R and SdhD-R47W substitutions

BACKGROUND: Succinate dehydrogenase inhibitor fungicides are important in the management of Zymoseptoria tritici in wheat. New active ingredients from this group of fungicides have been introduced recently and are widely used. Because the fungicides act at a single enzyme site, resistance development in Z. tritici is classified as medium-to-high risk. RESULTS: Isolates from Irish experimental plots in 2015 were tested against the SDHI penthiopyrad during routine monitoring. The median of the population was approximately 2 x less sensitive than the median of the baseline population. Two of the 93 isolates were much less sensitive to penthiopyrad than least sensitive of the baseline isolates. These isolates were also insensitive to most of commercially available SDHIs. Analysis of the succinate dehydrogenase coding genes confirmed the presence of the substitutions SdhC-H152R and SdhD-R47W in the very insensitive isolates. CONCLUSION: This is the first report showing that the SdhC-H152R mutation detected in laboratory mutagenesis studies also exists in the field. The function and relevance of this mutation, combined with SdhD-R47W, still needs to be determined.

Exercise-stimulated GLUT4 Expression is Similar in Normotensive and Hypertensive Rats

The effects of exercise training on systolic blood pressure (BP), insulin sensitivity, and plasma membrane GLUT4 protein content in spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats were compared. 16 SHR and 16 WKY male rats, aged 6 months, were randomized into sedentary and trained (tread-mill running, 5 days/week, 60 min/day for 10 weeks) groups (n = 8/group). At baseline, SHR had lower insulin sensitivity than WKY rats, however, there were no differences between WKY and SHR GLUT4 expression. The 10-week training reduced BP by similar to 19% in SHR, improved insulin sensitivity by similar to 24% in SHR, but not in WKY, and increased GLUT4 expression in both animal models. Compared to the sedentary group, there was an increase of GLUT4 in WKY rats by similar to 25% in the heart, by similar to 23% in the gastrocnemius, and by similar to 15% in the fat tissue. Trained SHR presented an increase in GLUT4 of similar to 21%, similar to 20%, and similar to 14%, in the same tissues, respectively. There were no differences between SHR and WKY rats in post-training GLUT4 expression. We conclude that training determined BP and insulin resistance reduction in SHR, and increased GLUT4 expression in both normotensive and hypertensive rats. However, considering the similar rise in GLUT4-induced training in SHR and WKY, it is possible that GLUT4 levels in plasma membrane fraction do not have a pivotal role in the exercise-induced improvement of insulin sensitivity in SHR.

VALLEY SPLITTING AND g-FACTOR IN AlAs QUANTUM WELLS

Here we present the results of magneto resistance measurements in tilted magnetic field and compare them with calculations. The comparison between calculated and measured spectra for the case of perpendicular fields enable us to estimate the dependence of the valley splitting as a function of the magnetic field and the total Lande g-factor (which is assumed to be independent of the magnetic field). Since both the exchange contribution to the Zeeman splitting as well as the valley splitting are properties associated with the 2D quantum confinement, they depend only on the perpendicular component of the magnetic field, while the bare Zeeman splitting depends on the total magnetic field. This information aided by the comparison between experimental and calculated gray scale maps permits to obtain separately the values of the exchange and the bare contribution to the g-factor.

Oroidin Inhibits the Activity of the Multidrug Resistance Target Pdr5p from Yeast Plasma Membranes

Oroidin was isolated from the marine sponge Agelas sventres and inhibited the activity and function of Pdr5p, an enzyme responsible for the multidrug resistance phenotype in Saccharomyces cerevisiae. This compound may help in the development of new drugs that reverse this dangerous phenotype of pathogenic yeast and fungi.

Salmoninae Commensal Bacteria Show a Positive Correlation Between Antibiotic Resistance and Mercury Resistance Profiles

Bacterial isolates from natural sites with high toxic and heavy metal contamination more frequently contain determinants for resistance to antimicrobials. Natural strains were isolated from the ingesta and external slime of Salmo salar (Linnaeus, 1758) and Salvelinusjontinalis (Mitchell, 1814). Fish specimens were acquired from Casco Bay hatcheries, Casco, ME where there is no history of antibiotic use. Seventy-nine bacterial strains, including many well-documented salmonid commensals (an association from which the fish derives no benefit), were identified using 165 rRNA gene sequencing. Mercury resistant isolates were selected for initially on 25μM HgCI2. Strains were then grown at 20-24°C on Trypticase Soy Agar (TSA) plates containing 0-1000μM HgCl2 or 0-130μM Phenyl Mercuric Acetate (PMA). Mercury in the hatchery feed water due to ubiquitous non-point source deposition has selected for the mercury resistance observed in bacterial strains. Antibiotic resistance determinations, as measured by Minimum Inhibitory Concentration MIC) assays were performed on the 79 bacterial isolates using Sensititrel antimicrobial susceptibility panels. A positive linear correlation between the mercury (pMA and HgCl2) MIC's and antibiotic resistance for all observed strains was demonstrated. Conjugation experiments with Pseudomonas, Aeromonas, and Azomonas donors confirmed phenotypic transfer of penicillin and cephem resistances to Escherichia coli DH5a recipients. Conjugation experiments with Pseudomonas donors showed minimal transfer of tetracycline and minoglycoside resistances to Escherichia coli DH5a recipients. Our study suggests that the accumulation of antimicrobial resistances observed in these natural bacterial populations may be due to the indirect selective pressure exerted by environmental mercury.

Nonpreference of whitefly for oviposition in tomato genotypes

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Restriction fragment length polymorphism (RFLP) in Exon 2 of the BoLA-DRB3 gene in South American cattle

The Bola-DRB3 gene participates in the development of the immune response and is highly polymorphic. For these reasons, it has been a candidate gene in studies of the genetic basis of disease resistance and in population genetic analysis. South American native cattle breeds have been widely replaced by improved exotic breeds leading to a loss of genetic resources. In particular South American native breeds have high levels of fertility and disease resistance. This work describes genetic variability in the BoLA-DRB3 gene in native (Caracu, Pantaneiro, Argentinean Creole) and exotic (Holstein, Jersey, Nelore, Gir) cattle breeds in Brazil and Argentina. PCR-RFLP alleles were identified by combining the restriction patterns for the BoLA-DRB3.2 locus obtained with RsaI, BstY, and HaeIII restriction enzymes. Allelic frequencies and deviations from the Hardy-Weinberg equilibrium were also calculated. Analysis of the 24 BoLA-DRB3 PCR-RFLP alleles identified showed differences in the allele distributions among breeds.

Enhanced natural killer activity and production of pro-inflammatory cytokines in mice selected for high acute inflammatory response (AIRmax)

Strains of mice with maximal and minimal acute inflammatory responsiveness (AIRmax and AIRmin, respectively) were developed through selective breeding based on their high- or low-acute inflammatory responsiveness. Previous reports have shown that AIRmax mice are more resistant to the development of a variety of tumours than AIRmin mice, including spontaneous metastasis of murine melanoma. Natural killer activity is involved in immunosurveillance against tumour development, so we analysed the number and activity of natural killer cells (CD49b(+)), T-lymphocyte subsets and in vitro cytokine production by spleen cells of normal AIRmax and AIRmin mice. Analysis of lymphocyte subsets by flow cytometry showed that AIRmax mice had a higher relative number of CD49b(+) cells than AIRmin mice, as well as cytolytic activity against Yac.1 target cells. The number of CD3(+) CD8(+) cells was also higher in AIRmax mice. These findings were associated with the ability of spleen cells from AIRmax mice in vitro to produce higher levels of the pro-inflammatory cytokines tumour necrosis factor-alpha, interleukin-12p40 and interferon-gamma but not the anti-inflammatory interleukin-10. Taken together, our data suggest that the selective breeding to achieve the AIRmax and AIRmin strains was able to polarize the genes associated with cytotoxic activity, which can be responsible for the antitumour resistance observed in AIRmax mice.