Aims: Quinolone antibiotics are the agents of choice for treating systemic Salmonella infections. Resistance to quinolones is usually mediated by mutations in the DNA gyrase gene gyrA. Here we report ...the evaluation of standard HPLC equipment for the detection of mutations (single nucleotide polymorphisms; SNPs) in gyrA, gyrB, parC and parE by denaturing high performance liquid chromatography (DHPLC). Methods: A panel of Salmonella strains was assembled which comprised those with known different mutations in gyrA (n = 8) and fluoroquinolone-susceptible and -resistant strains (n = 50) that had not been tested for mutations in gyrA. Additionally, antibiotic-susceptible strains of serotypes other than Salmonella enterica serovar Typhimurium strains were examined for serotype-specific mutations in gyrB (n = 4), parC (n = 6) and parE (n = 1). Wild-type (WT) control DNA was prepared from Salmonella Typhimurium NCTC 74. The DNA of respective strains was amplified by PCR using Optimase® proofreading DNA polymerase. Duplex DNA samples were analysed using an Agilent A1100 HPLC system with a Varian Helix™ DNA column. Sequencing was used to validate mutations detected by DHPLC in the strains with unknown mutations. Results: Using this HPLC system, mutations in gyrA, gyrB, parC and parE were readily detected by comparison with control chromatograms. Sequencing confirmed the gyrA predicted mutations as detected by DHPLC in the unknown strains and also confirmed serotype-associated sequence changes in non-Typhimurium serotypes. Conclusions: The results demonstrated that a non-specialist standard HPLC machine fitted with a generally available column can be used to detect SNPs in gyrA, gyrB, parC and parE genes by DHPLC. Wider applications should be possible.
Aims: In view of recent findings that a multidrug efflux pump CmeABC exists in Campylobacter jejuni, 391 C. jejuni and 52 Campylobacter coli of human and animal origin were examined for a multidrug ...resistance phenotype. Materials and methods: The MICs of ampicillin, chloramphenicol, ciprofloxacin, erythromycin, kanamycin, tetracycline, cetrimide, triclosan, acridine orange, paraquat and ethidium bromide were determined. Resistance to organic solvents and the effect of salicylate (known inducer of the marRAB operon in Escherichia coli and Salmonella) were also examined. Results: Two C. coli and 13 C. jejuni isolates, mainly from pigs or poultry, were resistant to three or more antibiotics and 12 of these strains had reduced susceptibility to acridine orange and/or ethidium bromide. Strains (n = 20) that were less susceptible to acridine orange, ethidium bromide and triclosan were significantly more resistant (P < 0.05) to ampicillin, chloramphenicol, ciprofloxacin, erythromycin, nalidixic acid and tetracycline, with two- to four-fold increases in MIC values compared with strains (n = 20) most susceptible to acridine orange, ethidium bromide and triclosan. Growth of strains with 1 mM salicylate caused a small (up to two-fold) but statistically significant (P ≤ 0.005) increase in the MICs of chloramphenicol, ciprofloxacin, erythromycin and tetracycline. Conclusions: These data indicate that multiple antibiotic resistant (MAR)-like Campylobacter strains occur and it may be postulated that these may overexpress cmeABC or another efflux system.
The relationship between DNA methylation and histone acetylation at the imprinted mouse genes U2af1-rs1 and Snrpn is explored by chromatin immunoprecipitation (ChIP) and resolution of parental ...alleles using single-strand conformational polymorphisms. The U2af1-rs1 gene lies within a differentially methylated region (DMR), while Snrpn has a 5' DMR (DMR1) with sequences homologous to the imprinting control center of the Prader-Willi/Angelman region. For both DMR1 of Snrpn and the 5' untranslated region (5'-UTR) and 3'-UTR of U2af1-rs1, the methylated and nonexpressed maternal allele was underacetylated, relative to the paternal allele, at all H3 lysines tested (K14, K9, and K18). For H4, underacetylation of the maternal allele was exclusively (U2af1-rs1) or predominantly (Snrpn) at lysine 5. Essentially the same patterns of differential acetylation were found in embryonic stem (ES) cells, embryo fibroblasts, and adult liver from F1 mice and in ES cells from mice that were dipaternal or dimaternal for U2af1-rs1. In contrast, in a region within Snrpn that has biallelic methylation in the cells and tissues analyzed, the paternal (expressed) allele showed relatively increased acetylation of H4 but not of H3. The methyl-CpG-binding-domain (MBD) protein MeCP2 was found, by ChIP, to be associated exclusively with the maternal U2af1-rs1 allele. To ask whether DNA methylation is associated with histone deacetylation, we produced mice with transgene-induced methylation at the paternal allele of U2af1-rs1. In these mice, H3 was underacetylated across both the parental U2af1-rs1 alleles whereas H4 acetylation was unaltered. Collectively, these data are consistent with the hypothesis that CpG methylation leads to deacetylation of histone H3, but not H4, through a process that involves selective binding of MBD proteins.
Enteric bacteria with resistance to third and fourth generation cephalosporin antibiotics, especially Escherichia coli bearing the blaCTX-M gene, have been detected in a wide range of food producing ...animals. However, commercial vaccines for these organisms are not currently available.
An autogenous vaccine was prepared from E. coli bearing the blaCTX-M-14 gene and evaluated as a potential control measure to reduce shedding and dissemination of these organisms in cattle. Calves (n=30) received either an autogenous vaccine prepared from E. coli serotype O33 bearing the blaCTX-M-14 gene or a placebo by intramuscular injection on three separate occasions. Two weeks after the final vaccination, all calves were challenged by oral gavage with the O33 CTX-M-14 strain of E. coli (1×1010 CFU). Faeces, intestinal mucosa and blood samples were taken for enumeration of total and CTX-M-14 E. coli and for assessment of the humoral immune response.
The cumulative number of total E. coli excreted at 7days post-challenge was significantly (p=0.006) lower in the vaccinated group than the placebo group. However, there was no significant difference in the shedding of either CTX-M-14 E. coli or total E. coli between vaccinated and placebo calves throughout the study period. The systemic immune response to E. coli O33 antigen was tested by ELISA and was significantly higher (p<0.001) in vaccinated than placebo calves. However, there was no significant difference in the mucosal immune response. These findings do not support the use of autogenous vaccination for the control of CTX-M-14 E. coli in calves.
Abstract
Recent attention has focused on accelerated glacial losses along the Amundsen Sea coast that result from changes in atmosphere and ocean circulation, with sea ice playing a mediating but not ...well-understood role. Here, we investigated how sea ice has changed in the Amundsen Sea over the period of 1979 to 2014, focusing on spatio-temporal changes in ice edge advance/retreat and percent sea ice cover in relation to changes in winds. In contrast to the widespread sea ice decreases to the east and increases to the west of the Amundsen Sea, sea ice changes in the Amundsen Sea were confined to three areas: (i) offshore of the shelf break, (ii) the southern Pine Island Polynya, and (iii) the eastern Amundsen Sea Polynya. Offshore, a 2-month decrease in ice season duration coincided with seasonal shifts in wind speed and direction from March to May (relating to later ice advance) and from September to August (relating to earlier retreat), consistent with reported changes in the depth/location of the Amundsen Sea Low. In contrast, sea ice decreases in the polynya areas corresponded to episodic or step changes in spring ice retreat (earlier by 1–2 months) and were coincident with changes to Thwaites Iceberg Tongue (located between the two polynyas) and increased southeasterly winds. Temporal correlations among these three areas were weak, indicating different local forcing and/or differential response to large-scale forcing. Although our analysis has shown that part of the variability can be explained by changes in winds or to the coastal icescape, an additional but unknown factor is how sea ice has responded to changes in ocean heat and freshwater inputs. Unraveling cause and effect, critical for predicting changes to this rapidly evolving ocean-ice shelf-sea ice system, will require in situ observations, along with improved remote sensing capabilities and ocean modeling.
Fluoroquinolones are a widely used group of antimicrobials in both human and animal medicine, with ciprofloxacin being a critically important fluoroquinolone for serious human infections. The present ...study reports on a 1-year survey for the presence of ciprofloxacin-resistant Escherichia coli in turkeys from Great Britain. Boot swabs were taken from 442 turkey flocks comprised of 125 breeding flocks and 317 meat flocks from 337 different farms over a 1-year period (2006 to 2007). CHROMagar ECC containing 1 mg/l ciprofloxacin was used to obtain ciprofloxacin-resistant isolates. Isolates were tested for sensitivity to 16 different antimicrobials. Isolates with ciprofloxacin minimum inhibitory concentrations ≥8 mg/l were tested for mutations in gyrA by polymerase chain reaction and DNA sequencing. Selected isolates were tested by multiplex polymerase chain reaction for qnrA, qnrB and qnrS, qepA and aac(6′)-Ib-cr genes. Conjugations were performed to assess the transferability of resistance to quinolones. Ciprofloxacin-resistant E. coli was found in 22.4% of turkey breeding flocks and 60.9% of meat flocks. Two main mutations in gyrA, as well as a range of silent mutations, were identified in resistant isolates. Flocks with transferable resistance genes qnrB, qnrS, and aac(6′)-Ib-cr were found at a low flock prevalence of 4.2%, 1.6% and 1.0%, respectively; however, under laboratory conditions only transfer of qnrS genes could be demonstrated. This work has confirmed the occurrence of ciprofloxacin-resistant E. coli strains throughout turkey breeding and meat flocks, with almost one-third of E. coli isolates being resistant to ciprofloxacin. Of those, more than 87% were also resistant to three or more antimicrobial classes.
We conducted playback experiments with wild bottlenose dolphins,Tursiops truncatus, to determine whether there is sufficient information in their individually distinctive signature whistles for ...individual recognition. We conducted experiments with members of a resident community of dolphins in waters near Sarasota, Florida, during temporary capture–release projects. We used a paired playback design, wherein the same two whistle sequences were predicted to evoke opposite responses from two different target animals. This design controlled for any unknown cues that may have been present in the playback stimuli. We predicted that mothers would respond more strongly to the whistles of their own independent offspring than to the whistles of a familiar, similar-aged nonoffspring. Similarly, we predicted that independent offspring would respond more strongly to the whistles of their own mother than to the whistles of a familiar, similar-aged female. Target animals were significantly (P<0.02) more likely to respond to the predicted stimuli, with responses measured by the number of head turns towards the playback speaker. In bottlenose dolphin societies, stable, individual-specific relationships are intermixed with fluid patterns of association between individuals. In primate species that live in similar ‘fission–fusion’ type societies, individual recognition is commonplace. Thus, when taken in the context of what is known about the social structure and behaviour of bottlenose dolphins, these playback experiments suggest that signature whistles are used for individual recognition.
An oxide/oxide ceramic fiber‐matrix composite (CMC) has been extensively characterized for high‐temperature aerospace structural applications. This CMC is called GEN‐IV™, and it has a porous and ...cracked aluminosilicate matrix reinforced by 3M Nextel 610™ alumina fibers woven in a balanced eight harness weave (8HSW). This CMC has been specifically designed without an interphase between the fiber and matrix, and it relies on the porous matrix for flaw tolerance. Stress‐strain response is nearly linear to failure and without a well‐defined proportional limit in tension and compression. In‐plane shear and interlaminar strength increases with increasing temperature. The 1000°C fatigue limit in air at 105 cycles is 160 MPa, and the residual tensile strength of run‐out specimens is not affected by the fatigue loading. The creep‐rupture resistance above 1000°C is relatively poor, but it can be improved with a more‐creep‐resistant fiber.
Surgery has been historically the preferred primary treatment for patients with well-differentiated thyroid carcinoma and for selected locoregional recurrences. Adjuvant therapy with radioactive ...iodine is typically recommended for patients with an intermediate to high risk of recurrence. Despite these treatments, locally advanced disease and locoregional relapses are not infrequent. These patients have a prolonged overall survival that may result in long periods of active disease and the possibility of requiring subsequent treatments. Recently, many new options have emerged as salvage therapies. This review offers a comprehensive discussion and considerations regarding surgery, active surveillance, radioactive iodine therapy, ultrasonography-guided percutaneous ablation, external beam radiotherapy, and systemic therapy for well-differentiated thyroid cancer based on relevant publications and current reference guidelines. We feel that the surgical member of the thyroid cancer management team is empowered by being aware and facile with all management options.
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