Profiles of phenotype resistance to antibiotic other than β-lactams in Klebsiella pneumoniae ESBLs-producers, carrying blaSHV genes

Extended spectrum β-lactamases production is one of the most common mechanism of resistance to extended spectrum β-lactam antibiotics is increasing worldwide. Twenty five strains of Klebsiella pneumoniae isolated from clinical specimens were tested. Based on the phenotypic confirmatory test all these strains were defined as ESBL producers named ESBL(+). The plasmid DNA from each strains was used to investigate the presence of blaSHV genes responsible for extended spectrum β-lactamases production. Moreover, susceptibility of these strains to antibiotic other than β-lactams in was tested.     

Characterization of a new β-glucosidase/β-xylosidase from the gut microbiota of the termite (Reticulitermes santonensis)

The gut of the termite Reticulitermes santonensis contains an interesting diversity of prokaryotic and eukaryotic microorganisms not found elsewhere. These microorganisms produce many enzyme-digesting lignocellulosic compounds, probably in cooperation with endogenous enzymes. Regarding cellulose and hemicellulose digestion in the termite gut, much remains to be learned about the relative contributions of termite enzymes and enzymes produced by different microorganisms. Here we grew bacterial colonies from termite gut suspensions, identifying 11 of them after PCR amplification of their 16S rRNA genes. After constructing in Escherichia coli a genomic DNA library corresponding to all of the colonies obtained, we performed functional screening for α-amylase, xylanase, β-glucosidase, and endoglucanase activities. This screen revealed a clone producing β-glucosidase activity. Sequence analysis showed that the cloned genomic DNA fragment contained three complete ORFs (bglG, bglF, and bglB) organized in a putative bgl operon. The new β-glucosidase (BglB), identified with its regulators BglG and BglF, belongs to glycoside hydrolase family 1. The new β-glucosidase was expressed in E. coli and purified by affinity chromatography. The purified enzyme shows maximal activity at pH 6.0 and 40?°C. It also displays β-xylosidase activity.     

Trends in antibiotic resistance in Prevotella species from patients of the University Hospital of Maxillofacial Surgery,Sofia, Bulgaria,in 2003–2009

Head-and-neck infections often involve anaerobes such as Prevotella species. Aim of the present study was to assess the evolution and the factors associated with resistance in Prevotella species to penicillin, clindamycin, metronidazole, tetracycline and β-lactams/β-lactamase inhibitors (BL/BLIs). In total, 192 Prevotella strains, isolated from patients with oral and head-and-neck infections, were evaluated. Common isolates were Prevotella intermedia and Prevotella melaninogenica within the pigmented species as well as Prevotella oris and Prevotella oralis group within the non-pigmented species. Overall resistance was 43.2% for penicillin, 10.9% for clindamycin, 0% for metronidazole. Nonsusceptibility to tetracycline was 29.1% without significant differences in resistance rates between pigmented and other species. Penicillin resistant strains were β-lactamase positive. From 2003–2004 to 2007–2009, penicillin resistance rates increased about four-fold (from 15.4% to 60.6%). Clindamycin resistance did not show evolution, whereas tetracycline nonsusceptibility decreased from 43.3% in 2003–2004 to 20.7% in 2007–2009. Except for one (0.5%) P. oralis strain with intermediate susceptibility to BL/BLIs, the other strains were susceptible to the agents. In conclusion, in Prevotella strains from patients with head-and-neck infections, the resistance rate to penicillin increased, that to clindamycin remained stable and the nonsusceptibility rate to tetracycline decreased during the period. Activity against >99% of Prevotella strains was observed with metronidazole and BL/BLIs. The penicillin resistance and tetracycline nonsusceptibility were associated with the year of study, national antibiotic consumption and possibly with previous treatment (for tetracycline). The evolution of penicillin resistance in Prevotella strains was highly dynamic.     

Identification,mapping, and marker development of stem rust resistance genes in durum wheat ‘Lebsock’

Wheat production in many wheat-growing regions is vulnerable to stem rust, caused by Puccinia graminis f. sp. tritici (Pgt). Several previous studies showed that most of the durum cultivars adapted to the upper Great Plains in the USA have good resistance to the major Pgt pathotypes, including the Ug99 race group. To identify the stem rust resistance (Sr) genes in the durum cultivar ‘Lebsock’, a tetraploid doubled haploid (DH) population derived from a cross between Lebsock and Triticum turgidum ssp. carthlicum PI 94749 was screened with the Pgt races TTKSK, TRTTF, and TTTTF. The stem rust data and the genotypic data previously developed were used to identify quantitative trait loci (QTL) associated with resistance. We identified one QTL each on chromosome arms 4AL, 6AS, 6AL, and 2BL. Based on marker and race-specification analysis, we postulated that the QTL on 4AL, 6AS, 6AL, and 2BL correspond to Sr7a, Sr8155B1, Sr13, and likely Sr9e, respectively. The results indicated that most of the US durum germplasm adapted to the upper Great Plains likely harbors the four major Sr genes characterized in this study. Among these genes, Sr8155B1 was recently identified and shown to be unique in that it conferred susceptibility to TTKSK but resistance to variant race TTKST. Two, three, and one thermal asymmetric reverse PCR (STARP) markers were developed for Sr7a, Sr8155-B1, and Sr13, respectively. Knowledge of the Sr genes in durum germplasm and the new STARP markers will be useful to pyramid and deploy multiple Sr genes in future durum and wheat cultivars.     

Trends in Malaria in Odisha,India—An Analysis of the 2003–2013 Time-Series Data from the National Vector Borne Disease Control Program

Background

Although Odisha is the largest contributor to the malaria burden in India, no systematic study has examined its malaria trends. Hence, the spatio-temporal trends in malaria in Odisha were assessed against the backdrop of the various anti-malaria strategies implemented in the state.

Methods

Using the district-wise malaria incidence and blood examination data (2003–2013) from the National Vector Borne Disease Control Program, blood examination-adjusted time-trends in malaria incidence were estimated and predicted for 2003–2013 and 2014–2016, respectively. An interrupted time series analysis using segmented regression was conducted to compare the disease trends between the pre (2003–2007) and post-intensification (2009–2013) periods. Key-informant interviews of state stakeholders were used to collect the information on the various anti-malaria strategies adopted in the state.

Results

The state annual malaria incidence declined from 10.82/1000 to 5.28/1000 during 2003–2013 (adjusted annual decline: -0.54/1000, 95% CI: -0.78 to -0.30). However, the annual blood examination rate remained almost unchanged from 11.25% to 11.77%. The keyinformants revealed that intensification of anti-malaria activities in 2008 led to a more rapid decline in malaria incidence during 2009–2013 as compared to that in 2003–2007 [adjusted decline: -0.83 (-1.30 to -0.37) and -0.27 (-0.41 to -0.13), respectively]. There was a significant difference in the two temporal slopes, i.e., -0.054 (-0.10 to -0.002, p = 0.04) per 1000 population per month, between these two periods, indicating almost a 200% greater decline in the post-intensification period. Although, the seven southern high-burden districts registered the highest decline, they continued to remain in that zone, thereby, making the achievement of malaria elimination (incidence <1/1000) unlikely by 2017.

Conclusion

The anti-malaria strategies in Odisha, especially their intensification since 2008, have helped improve its malaria situation in recent years. These successful measures need to be sustained and perhaps intensified further for eliminating malaria from Odisha.     

Role of phages in the pathogenesis of Burkholderia,or ‘Where are the toxin genes in Burkholderia phages?’

Most bacteria of the genus Burkholderia are soil- and rhizosphere-associated, and rhizosphere associated, noted for their metabolic plasticity in the utilization of a wide range of organic compounds as carbon sources. Many Burkholderia species are also opportunistic human and plant pathogens, and the distinction between environmental, plant, and human pathogens is not always clear. Burkholderia phages are not uncommon and multiple cryptic prophages are identifiable in the sequenced Burkholderia genomes. Phages have played a crucial role in the transmission of virulence factors among many important pathogens; however, the data do not yet support a significant correlation between phages and pathogenicity in the Burkholderia. This may be due to the role of Burkholderia as a 'versaphile' such that selection is occurring in several niches, including as a pathogen and in the context of environmental survival.     

‘To be,or not to be’—The dilemma of ‘silent’ antimicrobial resistance genes in bacteria

Antimicrobial resistance is a serious threat to public health that dramatically undermines our ability to treat bacterial infections. Microorganisms exhibit resistance to different drug classes by acquiring resistance determinants through multiple mechanisms including horizontal gene transfer. The presence of drug resistance genotypes is mostly associated with corresponding phenotypic resistance against the particular antibiotic. However, bacterial communities harbouring silent antimicrobial resistance genes—genes whose presence is not associated with a corresponding resistant phenotype do exist. Under suitable conditions, the expression pattern of such genes often revert and regain resistance and could potentially lead to therapeutic failure. We often miss the presence of silent genes, since the current experimental paradigms are focused on resistant strains. Therefore, the knowledge on the prevalence, importance and mechanism of silent antibiotic resistance genes in bacterial pathogens are very limited. Silent genes, therefore, provide an additional level of complexity in the war against drug-resistant bacteria, reminding us that not only phenotypically resistant strains but also susceptible strains should be carefully investigated. In this review, we discuss the presence of silent antimicrobial resistance genes in bacteria, their relevance and their importance in public health.     

Characterization of VanY(n) , a novel d,d-peptidase/d,d-carboxypeptidase involved in glycopeptide antibiotic resistance in Nonomuraea sp. ATCC 39727

VanY(n) is a novel protein involved in the mechanism of self-resistance in Nonomuraea sp. ATCC?39727, which produces the glycopeptide antibiotic A40926, the precursor of the second-generation dalbavancin, which is in phase?III of clinical development. VanY(n) (196 residues) is encoded by the dbv7 gene within the dbv biosynthetic cluster devoted to A40926 production. C-terminal His6-tagged VanY(n) was successfully expressed as a soluble and active protein in Escherichia?coli. The analysis of the sequence suggests the presence of a hydrophobic transmembrane portion and two conserved sequences (SxHxxGxAxD and ExxH) in the extracytoplasmic domain that are potentially involved in coordination of Zn(2+) and catalytic activity. The presence of these conserved sequences indicates a similar mechanism of action and substrate binding in VanY(n) as in VanY, VanX and VanXY Zn(2+) -dependent d,d-carboxypeptidases and d-Ala-d-Ala dipeptidases acting on peptidoglycan maturation and involved in glycopeptide resistance in pathogens. On substrates mimicking peptidoglycan precursors, VanY(n) shows d,d-carboxypeptidase and d,d-dipeptidase activity, but lacks d,d-carboxyesterase ability on d-Ala-d-Lac-terminating peptides. VanY(n) belongs to the metallo-d,d-carboxypeptidase family, but it is inhibited by β-lactams. Its characterization provides new insights into the evolution and transfer of resistance determinants from environmental glycopeptide-producing actinomycetes (such as Nonomuraea sp.) to glycopeptide-resistant pathogens (enterococci and staphylococci). It may also contribute to an early warning system for emerging resistance mechanisms following the introduction into clinics of a second-generation glycopeptide such as dalbavancin. Database The nucleotide sequence of vanY(n) is available in the GenBank data base under accession number CAD91202.     

Distribution, abundance, and utilization of wild berries by the gwich’in people in the Mackenzie River Delta region

This paper examines the abundance and Aboriginal use of botanical non-timber forest products in the Gwich’in Settlement Area in the Northwest Territories, Canada. Informants indicated that wild berries are important to Gwich’in diets and an important factor in culture and traditional land use. People collected mostly cloudberries (Rubus chamaemorus), blueberries (Vaccinium uliginosum), and lingonberries (Vaccinium vitisidaea). The total quantities collected were estimated to be greater than other published accounts of berry gathering by northern Aboriginal peoples in North America. The total collected by the 450 households in the year 2000 was about 16,000 L. Inventories of wild berries on the landscape suggest that substantial quantities could be collected for commercial production, although the between-year supply can show considerable variation.     

Marker-assisted dissection of the oligogenic anthracnose resistance in the common bean cultivar, ‘G2333’

 Two independently assorting dominant genes conditioning resistance to bean anthracnose were identified in an F₂ population derived from the highly resistant bean differential cultivar, ‘G 2333’. One gene was allelic to the Co-4 gene in the differential cultivar ‘TO’ and was named Co-4 ² , whereas the second gene was assigned the temporary name Co-7 until a complete characterization with other known resistance genes can be conducted. Two RAPD markers linked to the Co-4 ² allele were identified. One RAPD, OAS13₉₅₀, co-segregated with no recombinants in two segregating populations of 143 F₂ individuals, whereas the second RAPD, OAL9₇₄₀, mapped at 3.9 cM from the Co-4 ² allele. Two 24-mer SCAR primers (SAS13), developed from the OAS13₉₅₀ RAPD marker, were dominant and polymorphic, similar to the original RAPD, and supported the tight linkage between the marker(s) and the Co-4 ² allele. The markers were present in germplasm with known resistance alleles at the Co-4 locus. The presence of the markers in two other differential cultivars not previously characterized and in four navy bean cultivars suggests the existence of a gene family for anthracnose resistance at or near the Co-4 locus. Since the Co-7 gene was present only in germplasm which also possessed the Co-4 ² and Co-5 genes, the SAS13 markers were used in combination with standard inoculation techniques to identify F₃ lines in which the Co-7 gene was homozygous and the Co-4 ² allele was absent. A similar strategy of marker-assisted dissection is proposed to identify resistant lines in which the Co-5 gene is absent and the Co-7 gene is present by selecting against the OAB3₄₅₀ marker, which has been shown previously to be linked to the Co-5 gene. These genes cannot be distinguished using traditional screening methods since all current races of the pathogen virulent to the Co-5 gene are avirulent to the Co-4 ² and Co-7 genes. We describe the use of molecular markers tightly linked to resistance genes to facilitate the identification of an uncharacterized resistance gene for which no discriminating race of the pathogen is known. Received: 22 March 1997 / Accepted: 15 July 1997     

PCR-based molecular characterization, phylogenetic analysis and secondary structure of the 28S rDNA of Thaparocleidus wallagonius (Monogenea: Dactylogyridae) �C the most primitive species of this genus from India

Species of the monogenean genus Thaparocleidus are specific to freshwater siluriform fish. The infection caused by these gill parasites are a major health problem to fish. But, to focus the control strategies of these parasites, first it is important to establish an accurate discrimination by molecular methods. In the present study, phylogenetic and structural analysis of 28S region of ribosomal DNA of T. wallagonius species collected from fish Wallago attu from Meerut (U.P.), India, was carried out. In the first step, we amplified, sequenced 28S region of ribosomal DNA of T. wallagonius to establish the phylogenetic relationship with other species of this genus. T. wallagonius found on gill filaments of fish W. attu, is the most primitive parasite of this genus from India, was unequivocally discriminate from other species of the same genus in this study. A secondary-structure model of the large subunit rDNA was also predicted using a combined comparative and thermodynamic approach. Molecular morphometric and phylogenetic relationship of T. wallagonius are discussed in detailed that based on molecular analysis using bioinformatic tools.     

The role of transforming growth factor (TGF)-β in modulating the immune response and fibrogenesis in the gut

Transforming growth factor (TGF)-β, a pleiotropic cytokine released by both immune and non-immune cells in the gut, exerts an important tolerogenic action by promoting regulatory T cell differentiation. TGF-β also enhances enterocyte migration and regulates extracellular matrix turnover, thereby playing a crucial role in tissue remodeling in the gut. In this review we describe the mechanisms by which abnormal TGF-β signaling impairs intestinal immune tolerance and tissue repair, thus predisposing to the onset of immune-mediated bowel disorders, such as inflammatory bowel disease and celiac disease. Additionally, we will discuss potential therapeutic strategies aiming at restoring physiologic TGF-β signaling in chronic intestinal diseases.     

Expression of multiple resistance genes enhances tolerance to environmental stressors in transgenic poplar (Populus × euramericana 'Guariento')

Commercial and non-commercial plants face a variety of environmental stressors that often cannot be controlled. In this study, transgenic hybrid poplar (Populus × euramericana 'Guariento') harboring five effector genes (vgb, SacB, JERF36, BtCry3A and OC-I) were subjected to drought, salinity, waterlogging and insect stressors in greenhouse or laboratory conditions. Field trials were also conducted to investigate long-term effects of transgenic trees on insects and salt tolerance in the transformants. In greenhouse studies, two transgenic lines D5-20 and D5-21 showed improved growth, as evidenced by greater height and basal diameter increments and total biomass relative to the control plants after drought or salt stress treatments. The improved tolerance to drought and salt was primarily attributed to greater instantaneous water use efficiency (WUEi) in the transgenic trees. The chlorophyll concentrations tended to be higher in the transgenic lines under drought or saline conditions. Transformed trees in drought conditions accumulated more fructan and proline and had increased Fv/Fm ratios (maximum quantum yield of photosystem II) under waterlogging stress. Insect-feeding assays in the laboratory revealed a higher total mortality rate and lower exuviation index of leaf beetle [Plagiodera versicolora (Laicharting)] larvae fed with D5-21 leaves, suggesting enhanced insect resistance in the transgenic poplar. In field trials, the dominance of targeted insects on 2-year-old D5-21 transgenic trees was substantially lower than that of the controls, indicating enhanced resistance to Coleoptera. The average height and DBH (diameter at breast height) of 2.5-year-old transgenic trees growing in naturally saline soil were 3.80% and 4.12% greater than those of the control trees, but these increases were not significant. These results suggested that multiple stress-resistance properties in important crop tree species could be simultaneously improved, although additional research is needed to fully understand the relationships between the altered phenotypes and the function of each transgene in multigene transformants.     

Identification and characterization of genes abnormally expressed in wing-deficient mutant (flügellos) of the silkworm,Bombyx mori

The wing-deficient mutant, flügellos (fl), of the silkworm lacks four wings in the pupa and the adult, due to aberrant wing morphogenesis during metamorphosis. To elucidate the mechanisms of wing-specific deficiencies in the fl mutant, we used mRNA differential display and identified five genes abnormally expressed in the fl wing discs. Northern blot and RT-PCR analyses revealed that four genes were overexpressed, but the fifth one was not transcribed in the fl wing discs. The expression level of ribosome-associated protein p40 in the fl wing discs was elevated approximately 10 times compared to the wild-type (WT) discs. Another overexpressed gene CB10 encodes a novel wing-specific protein with a putative zinc-finger motif. Overexpression of two components of extracellular matrix, cuticle protein 18 (BMCP18) and a fibrillin-like protein AD10, may result in the abnormal wing morphogenesis in the fl mutant. In contrast, a novel member of multifunctional Ca2+-binding protein annexins, designated as annexin b13 (Anx b13), was expressed dominantly in the wing discs of WT but completely repressed in the fl tissues. Strong expression of Anx b13 in wing discs during the fourth and fifth instar indicates that ANX B13 plays an important role in wing morphogenesis.     

Eukaryotic microbiota in the surface waters and sea ice of the Southern Ocean: aspects of physiology, ecology and biodiversity in a ‘two-phase’ ecosystem

The Southern Ocean provides a habitat for microplankton which is strongly influenced by physical factors. Of these, one of the most important and striking is the formation of sea ice. Organisms in the ice form a unique community with specific properties and adaptations. Material and organisms are exchanged between the water column and the ice during the annual cycle, and ice is an important factor in modifying biogeochemical processes and exchange between ocean and atmosphere. The coupled system, in which a range of organisms alternate between a fluid and a solid medium, provides an interesting exercise in community ecology, and has implications for the assessment of biodiversity in understanding large-scale change.     

Pyramiding the antimicrobial PR1aCB and AATCB genes in ‘Tarocco’ blood orange (Citrus sinensis Osbeck) to enhance citrus canker resistance

Transgenic Research - Citrus canker, caused by Xanthomonas citri subsp. citri (Xcc), is a major bacterial disease responsible for substantial economic losses in citrus-producing areas. To breed...