Electron spin relaxation of P1 centers in synthetic diamonds with potential as B1 standards for DNP enhanced NMR.

The microwave magnetic field, B1, in the non-resonant structures typically used for DNP-enhanced NMR is relatively small, so calibration via continuous wave (CW) power saturation requires a sample with longer spin lattice relaxation times than the samples used as CW standards in X-band cavities. HPHT diamonds have strong, easily observed EPR signals from P1 centers (nitrogen defects), and are indefinitely stable. This makes HPHT diamonds attractive as secondary standards for calibration of electron B1 field strength in a variety of experimental arrangements. The concentrations of P1 centers is also typically in the 30-200 ppm range, or equivalently 10-60 mM, and therefore the EPR relaxation observed is relevant to DNP enhanced NMR employing free radical polarizing agents at similar concentrations. Pulsed and CW saturation relaxation measurements T1 and T2 are compared at X-band. Under CW conditions the relevant T1T2 product of time constants in our samples at room temperature is found to be dominated by electron-electron spin diffusion, and the product is large enough that saturation will be possible with the B1 of typical DNP systems. The similarity of T1 and T2 values obtained by pulse measurements at X-band and Q-band suggests that the X-band results can be extrapolated to the higher EPR frequencies used for DNP experiments. The electron spin dynamics observed here in HPHT diamond samples identify them as useful model systems to better delineate the interplay of electron spin relaxation, magic angle spinning, and inhomogeneous microwave irradiation as they affect DNP enhancement.

Evaluation of molecular and culture methods to determine the optimum testing strategy for verotoxigenic Escherichia coli in faecal specimens.

We evaluated 5 methods for the detection of verotoxogenic Escherichia coli (VTEC) from faecal specimens to determine the most sensitive and specific method(s) and to advise an optimum testing strategy. A total of 681 stool specimens were examined using up to 5 diagnostic molecular and phenotypic methods that are used routinely in the VTEC Reference laboratory, Dublin. A testing strategy incorporating a 2-step approach that included a single Real Time-PCR and 1 culture-based method yielded the highest sensitivity, specificity, positive predictive value, and negative predictive value of 98.21%, 100%, 100%, and 99.43%, respectively.

Extending high-finesse cavity techniques to the far-infrared.

Sensitive spectroscopic techniques involving high-finesse Fabry-Perot resonators are widely used in the microwave and near-infrared spectral regimes, but hardware limitations have hindered their extension to far-infrared wavelengths. While there is no theoretical limit to the frequency region where cavity-enhanced techniques are practical, the sensitivity of these methods does depend explicitly on the availability of highly reflective optics and, in the case of cavity ringdown spectroscopy, sufficiently fast detectors. Here, we describe a novel high-finesse cavity that uses wire-grid polarizers as the reflective surfaces. Quality factors on the order of 10(5) are achieved at 250 GHz. Based on the optimized cavity design, we investigate the feasibility of extending the cavity ringdown technique to far-infrared wavelengths. With the present commercially available technology, we find spectrometer performance to be limited by both the available optics and detectors. With a 120 cm cavity and a detector response time of ~500 ns, we predict a minimum detectable absorption coefficient, αmin, on the order of 10(-7) cm(-1). Given the sensitivity and noise requirements for the ringdown measurements, faster and more sensitive detectors are needed before implementation of the spectrometer is practical or offers any significant advances to existing methods at far-infrared wavelengths.

Characterization of farm, food, and clinical Shiga toxin-producing Escherichia coli (STEC) O113.

Thirty-nine Shiga toxin-producing Escherichia coli (STEC) O113 Irish farm, abattoir, and clinical isolates were analyzed in conjunction with eight Australian, New Zealand, and Norwegian strains for H (flagellar) antigens, virulence gene profile (eaeA, hlyA, tir, espA, espB katP, espP, etpD, saa, sab, toxB, iha, lpfA(O157/OI-141,) lpfA(O113,) and lpfA(O157/OI-154)), Shiga toxin gene variants (stx(1c), stx(1d), stx(2), stx(2c), stx(2dact), stx(2e), stx(2f,) and stx(2g)) and were genotyped using pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). All of the Irish strains were O113:H4, regardless of source, while all non-Irish isolates carried the H21 flagellar antigen. The stx(1) gene was present in 30 O113:H4 strains only, whereas the stx(2d) gene was common to all isolates regardless of source. In contrast, eaeA was absent, while hlyA was found in the Australian, New Zealand, Norwegian, and two of the Irish human clinical isolates. saa was present in the O113:H21 but not in the O113:H4 serotype. To the best of the author's knowledge, this is the first report of clinically significant STEC lacking both the eaeA and saa genes. PFGE analysis was inconclusive; however, MLST grouped the strains into three sequence types (ST): ST10, ST56, and ST223. Based on our findings, it was concluded that the stx(2d) gene is common in STEC O113, which are generally eaeA negative. Furthermore, STEC O113:H4 is a new, emerging bovine serotype of human clinical significance.

Multilocus sequence typing methods for the emerging Campylobacter Species C. hyointestinalis, C. lanienae, C. sputorum, C. concisus, and C. curvus.

Multilocus sequence typing (MLST) systems have been reported previously for multiple food- and food animal-associated Campylobacter species (e.g., C. jejuni, C. coli, C. lari, and C. fetus) to both differentiate strains and identify clonal lineages. These MLST methods focused primarily on campylobacters of human clinical (e.g., C. jejuni) or veterinary (e.g., C. fetus) relevance. However, other, emerging, Campylobacter species have been isolated increasingly from environmental, food animal, or human clinical samples. We describe herein four MLST methods for five emerging Campylobacter species: C. hyointestinalis, C. lanienae, C. sputorum, C. concisus, and C. curvus. The concisus/curvus method uses the loci aspA, atpA, glnA, gltA, glyA, ilvD, and pgm, whereas the other methods use the seven loci defined for C. jejuni (i.e., aspA, atpA, glnA, gltA, glyA, pgm, and tkt). Multiple food animal and human clinical C. hyointestinalis (n = 48), C. lanienae (n = 34), and C. sputorum (n = 24) isolates were typed, along with 86 human clinical C. concisus and C. curvus isolates. A large number of sequence types were identified using all four MLST methods. Additionally, these methods speciated unequivocally isolates that had been typed ambiguously using other molecular-based speciation methods, such as 16S rDNA sequencing. Finally, the design of degenerate primer pairs for some methods permitted the typing of related species; for example, the C. hyointestinalis primer pairs could be used to type C. fetus strains. Therefore, these novel Campylobacter MLST methods will prove useful in differentiating strains of multiple, emerging Campylobacter species.

Laboratory-based surveillance of human verocytotoxigenic Escherichia coli infection in the Republic of Ireland, 2002-2004.

The aim of this study was to examine the frequency and distribution of human verocytotoxigenic Escherichia coli (VTEC) O157 and non-O157 in the Republic of Ireland, and also to examine the presence of virulence genes in these isolates. This genetic information combined with phenotypic tests was used to produce a complete laboratory-based surveillance of human clinical VTEC infection in the Republic of Ireland between 2002 and 2004. Between January 2002 and December 2004 a total of 207 VTEC isolates were studied (one isolate per patient), 185 (89 %) of these were E. coli O157. The remaining 22 (11 %) were non-O157 E. coli, made up of 15 (7.2 %) E. coli O26, one (0.5 %) E. coli O103, one (0.5 %) E. coli O146, one (0.5 %) E. coli O145, two (1 %) E. coli O111 and two (1 %) ungroupable VTEC. These isolates originated from the eight health boards in the Republic of Ireland and represented over 90 % of the clinical cases of VTEC in the Republic of Ireland during this period. The results showed that VTEC O157 was the predominant serogroup and had a predominant toxin genotype of VT2 alone. Phage type 32 was the most common phage type of E. coli O157 identified. Non-O157 VTEC was a small proportion of all VTEC (10 % in 2002, 8 % in 2003, 15.5 % in 2004). In 2004 it was noted that there was an increase in the number and variety of non-O157 VTEC strains; however, this requires further monitoring in the future to see if this trend is sustained. It was also noted throughout the study period that the incidence of VTEC was higher in rural areas. Implementation of real-time PCR for the detection and subtyping of VTEC has aided outbreak investigations and is important for enhanced surveillance of VTEC in the Republic of Ireland.