Prevalence of mutations within the quinolone resistance-determining region of gyrA, gyrB, parC, and parE and association with antibiotic resistance in quinolone-resistant Salmonella enterica

Salmonella enterica isolates (n = 182) were examined for mutations in the quinolone resistance-determining region of gyrA, gyrB, parC, and parE. The frequency, location, and type of GyrA substitution varied with the serovar. Mutations were found in parC that encoded Thr57-Ser, Thr66-Ile, and Ser80-Arg substitutions. Mutations in the gyrB quinolone resistance-determining region were located at codon Tyr420-Cys or Arg437-Len. Novel mutations were also found in parE encoding Glu453-Gly, His461-Tyr, Ala498-Thr, Val512-Gly, and Ser518-Cys. Although it is counterintuitive, isolates with a mutation in both gyrA and parC were more susceptible to ciprofloxacin than were isolates with a mutation in gyrA alone.

Detection of gyrA mutations in quinolone-resistant Salmonella enterica by denaturing high-performance liquid chromatography

Denaturing high-performance liquid chromatography (DHPLC) was evaluated as a rapid screening and identification method for DNA sequence variation detection in the quinolone resistance-determining region of gyrA from Salmonella serovars. A total of 203 isolates of Salmonella were screened using this method. DHPLC analysis of 14 isolates representing each type of novel or multiple mutations and the wild type were compared with LightCycler-based PCR-gyrA hybridization mutation assay (GAMA) and single-strand conformational polymorphism (SSCP) analyses. The 14 isolates gave seven different SSCP patterns, and LightCycler detected four different mutations. DHPLC detected 11 DNA sequence variants at eight different codons, including those detected by LightCycler or SSCP. One of these mutations was silent. Five isolates contained multiple mutations, and four of these could be distinguished from the composite sequence variants by their DHPLC profile. Seven novel mutations were identified at five different loci not previously described in quinolone-resistant salmonella. DHPLC analysis proved advantageous for the detection of novel and multiple mutations. DHPLC also provides a rapid, high-throughput alternative to LightCycler and SSCP for screening frequently occurring mutations.

Emergence of fluoroquinolone resistance in the native Campylobacter coli population of pigs exposed to enrofloxacin

Objective: The effect of a single 5 day enrofloxacin treatment on the native Campylobacter coli population in conventionally weaned 5-week-old pigs was investigated. Materials: Twelve pigs were split into two groups of six: one group was treated with a therapeutic dose (15 mg/pig/day) of enrofloxacin and the other remained untreated to act as the control. Campylobacter coli were isolated from faecal samples and tested for ciprofloxacin resistance by measuring MIC values. Mutations in the quinolone resistance-determining region (QRDR) of the gyrA gene of resistant isolates were identified by sequencing and denaturing HPLC. Levels of enrofloxacin and its primary metabolite ciprofloxacin in the pig faeces were also measured by HPLC. Results: No quinolone-resistant C. coli (n = 867) were detected in any of the pigs prior to treatment, indicating <0.1% resistance in the group. Resistant C. coli were isolated from pigs for up to 35 days after treatment with a therapeutic dose. These resistant C. coli had MIC values of 128 mg/L and 8-16 mg/L for nalidixic acid and ciprofloxacin, respectively, and the same single point mutation causing a Thr-86 to Ile substitution in the QRDR was identified in each. The concentration of enrofloxacin in the pig faeces was 2-4 mug/g faeces for the duration of the 5 day therapeutic treatment and was detected up to 10 days post-treatment. Ciprofloxacin was also measured and peaked at 0.6 mug/g faeces in the treated group. Conclusion: This study provides evidence that a single course of enrofloxacin treatment contributes directly to the emergence and persistence of fluoroquinolone resistance in C. coli.

Evidence for multiple-antibiotic resistance in Campylobacter jejuni not mediated by CmeB or CmeF

An efflux system, CmeABC, in Campylobacter jejuni was previously described, and a second efflux system, CmeDEF, has now been identified. The substrates of CmeDEF include ampicillin, ethidium bromide, acridine, sodium dodecyl sulfate (SDS), deoxycholate, triclosan, and cetrimide, but not ciprofloxacin or erythromycin. C. jejuni NCTC11168 and two efflux pump knockout strains, cmeB::Kan(r) and cmeF::Kan(r), were exposed to 0.5 to 1 mu g of ciprofloxacin/ml in agar plates. All mutants arising from NCTC11168 were resistant to ciprofloxacin but not to other agents and contained a mutation resulting in the replacement of threonine 86 with isoleucine in the quinolone resistance-determining region of GyrA. Mutants with two distinct phenotypes were selected from the efflux pump knockout strains. Mutants with the first phenotype were resistant to ciprofloxacin only and had the same substitution within GyrA as the NCTC11168-derived mutants. Irrespective of the parent strain, mutants with the second phenotype were resistant to ciprofloxacin, chloramphenicol, tetracycline, ethidium bromide, acridine orange, and SDS and had no mutation in gyrA. These mutants expressed levels of the efflux pump genes cmeB and cmeF and the major outer membrane protein gene porA similar to those expressed by the respective parent strains. No mutations were detected in cmeF or cmeB. Accumulation assays revealed that the mutants accumulated lower concentrations of drug. These data suggest the involvement of a non-CmeB or -CmeF efflux pump or reduced uptake conferring multiple-antibiotic resistance, which can be selected after exposure to a fluoroquinolone.

The CtsR regulator of Listeria monocytogenes contains a variant glycine repeat region that affects piezotolerance, stress resistance, motility and virulence

A spontaneous high hydrostatic pressure (HHP)-tolerant mutant of Listeria monocytogenes ScottA, named AK01, was isolated previously. This mutant was immotile and showed increased resistance to heat, acid and H2O2 compared with the wild type (wt) (Karatzas, K.A.G. and Bennik, M.H.J. 2002 Appl Environ Microbiol 68: 3183–3189). In this study, we conclusively linked the increased HHP and stress tolerance of strain AK01 to a single codon deletion in ctsR (class three stress gene repressor) in a region encoding a highly conserved glycine repeat. CtsR negatively regulates the expression of the clp genes, including clpP, clpE and the clpC operon (encompassing ctsR itself), which belong to the class III heat shock genes. Allelic replacement of the ctsR gene in the wt background with the mutant ctsR gene, designated ctsRΔGly, rendered mutants with phenotypes and protein expression profiles identical to those of strain AK01. The expression levels of CtsR, ClpC and ClpP proteins were significantly higher in ctsRΔGly mutants than in the wt strain, indicative of the CtsRΔGly protein being inactive. Further evidence that the CtsRΔGly protein lacks its repressor function came from the finding that the Clp proteins in the mutant were not further induced upon heat shock, and that HHP tolerance of a ctsR deletion strain was as high as that of a ctsRΔGly mutant. The high HHP tolerance possibly results from the increased expression of the clp genes in the absence of (active) CtsR repressor. Importantly, the strains expressing CtsRΔGly show significantly attenuated virulence compared with the wt strain; however, no indication of disregulation of PrfA in the mutant strains was found. Our data highlight an important regulatory role of the glycine-rich region of CtsR in stress resistance and virulence.

Spatial and temporal scale issues in determining biomass burning regimes in Bolivia and Peru

ATSR-2 active fire data from 1996 to 2000, TRMM VIRS fire counts from 1998 to 2000 and burn scars derived from SPOT VEGETATION ( the Global Burnt Area 2000 product) were mapped for Peru and Bolivia to analyse the spatial distribution of burning and its intra- and inter-annual variability. The fire season in the region mainly occurs between May and October; though some variation was found between the six broad habitat types analysed: desert, grassland, savanna, dry forest, moist forest and yungas (the forested valleys on the eastern slope of the Andes). Increased levels of burning were generally recorded in ATSR-2 and TRMM VIRS fire data in response to the 1997/1998 El Nino, but in some areas the El Nino effect was masked by the more marked influences of socio-economic change on land use and land cover. There were differences between the three global datasets: ATSR-2 under-recorded fires in ecosystems with low net primary productivities. This was because fires are set during the day in this region and, when fuel loads are low, burn out before the ATSR-2 overpass in the region which is between 02.45 h and 03.30 h. TRMM VIRS was able to detect these fires because its overpasses cover the entire diurnal range on a monthly basis. The GBA2000 product has significant errors of commission (particularly areas of shadow in the well-dissected eastern Andes) and omission (in the agricultural zone around Santa Cruz, Bolivia and in north-west Peru). Particular attention was paid to biomass burning in high-altitude grasslands, where fire is an important pastoral management technique. Fires and burn scars from Landsat Thematic Mapper (TM) and Enhanced Thematic Mapper (ETM) data for a range of years between 1987 and 2000 were mapped for areas around Parque Nacional Rio Abiseo (Peru) and Parque Nacional Carrasco (Bolivia). Burn scars mapped in the grasslands of these two areas indicate far more burning had taken place than either the fires or the burn scars derived from global datasets. Mean scar sizes are smaller and have a smaller range in size between years the in the study area in Peru (6.6-7.1 ha) than Bolivia (16.9-162.5 ha). Trends in biomass burning in the two highland areas can be explained in terms of the changing socio-economic environments and impacts of conservation. The mismatch between the spatial scale of biomass burning in the high-altitude grasslands and the sensors used to derive global fire products means that an entire component of the fire regime in the region studied is omitted, despite its importance in the farming systems on the Andes.

Relationship between resistance factors and treatment efficacy when bromadiolone was used against anticoagulant-resistant Norway rats (Rattus norvegicus Berk.) in Wales

We investigated the relationship between the severity and incidence of resistance among Norway rats (Rattus norvegicus) on a farm in Wales and the subsequent outcome of a practical rodent control operation. Bromadiolone resistance factors were estimated for rats trapped on the farm using the blood clotting response test, and were found to be 2 to 3 for male rats and approximately 6 for females. The incidence of resistance in the rat population was high. Infestation size was estimated by census baiting and tracking, and was found to be substantial, with a maximum of 6.5 kg of bait being eaten on a single night. A proprietary rodenticide (Deadline (TM)), containing 0.005% bromadiolone, was used to control the infestation. The duration of baiting was 35 days and, according to the two methods of assessment used, treatment success was in the region of 87 and 93%. No evidence was observed of a significant impact of resistance on the rat control operation, and the remaining rats of this very heavy infestation would probably have been controlled if baiting had continued for longer.

Another look at the relative importance of sectors and regions in determining property returns

This paper re-examines the relative importance of sector and regional effects in determining property returns. Using the largest property database currently available in the world, we decompose the returns on individual properties into a national effect, common to all properties, and a number of sector and regional factors. However, unlike previous studies, we categorise the individual property data into an ever-increasing number of property-types and regions, from a simple 3-by-3 classification, up to a 10 by 63 sector/region classification. In this way we can test the impact that a finer classification has on the sector and regional effects. We confirm the earlier findings of previous studies that sector-specific effects have a greater influence on property returns than regional effects. We also find that the impact of the sector effect is robust across different classifications of sectors and regions. Nonetheless, the more refined sector and regional partitions uncover some interesting sector and regional differences, which were obscured in previous studies. All of which has important implications for property portfolio construction and analysis.

High prevalence of multidrug-tolerant bacteria and associated antimicrobial resistance genes isolated from ornamental fish and their carriage water

Background: Antimicrobials are used to directly control bacterial infections in pet (ornamental) fish and are routinely added to the water these fish are shipped in to suppress the growth of potential pathogens during transport. Methodology/Principal Findings: To assess the potential effects of this sustained selection pressure, 127 Aeromonas spp. isolated from warm and cold water ornamental fish species were screened for tolerance to 34 antimicrobials. Representative isolates were also examined for the presence of 54 resistance genes by a combination of miniaturized microarray and conventional PCR. Forty-seven of 94 Aeromonas spp. isolates recovered from tropical ornamental fish and their carriage water were tolerant to >= 15 antibiotics, representing seven or more different classes of antimicrobial. The quinolone and fluoroquinolone resistance gene, qnrS2, was detected at high frequency (37% tested recent isolates were positive by PCR). Class 1 integrons, IncA/C broad host range plasmids and a range of other antibiotic resistance genes, including floR, blaTEM21, tet(A), tet(D), tet(E), qacE2, sul1, and a number of different dihydrofolate reductase and aminoglycoside transferase coding genes were also detected in carriage water samples and bacterial isolates. Conclusions: These data suggest that ornamental fish and their carriage water act as a reservoir for both multi-resistant bacteria and resistance genes.

Antibiotic resistance genes, integrons and multiple antibiotic resistance in thirty-five serotypes of Salmonella enterica isolated from humans and animals in the UK

Objectives: To examine 397 strains of Salmonella enterica of human and animal origin comprising 35 serotypes for the presence of aadB, aphAI-IAB, aadA1, aadA2, bla(Carb(2)) or pse1, bla(Tem), cat1, cat2, dhfr1, floR, strA, sul1, sul2, tetA(A), tetA(B) and tetA(G) genes, the presence of class 1 integrons and the relationship of resistance genes to integrons and antibiotic resistance. Results: Some strains were resistant to ampicillin (91), chloramphenicol (85), gentamicin (2), kanamycin (14), spectinomycin (81), streptomycin (119), sulfadiazine (127), tetracycline (108) and trimethoprim (45); 219 strains were susceptible to all antibiotics. bla(Carb(2)), floR and tetA(G) genes were found in S. Typhimurium isolates and one strain of S. Emek only. Class 1 integrons were found in S. Emek, Haifa, Heidelberg, Mbandaka, Newport, Ohio, Stanley, Virchow and in Typhimurium, mainly phage types DT104 and U302. These strains were generally multi-resistant to up to seven antibiotics. Resistance to between three and six antibiotics was also associated with class 1 integron-negative strains of S. Binza, Dublin, Enteritidis, Hadar, Manhattan, Mbandaka, Montevideo, Newport, Typhimurium DT193 and Virchow. Conclusion: The results illustrate specificity of some resistance genes to S. Typhimurium or non- S. Typhimurium serotypes and the involvement of both class 1 integron and non-class 1 integron associated multi-resistance in several serotypes. These data also indicate that the bla(Carb(2)), floR and tetA(G) genes reported in the SG1 region of S. Typhimurium DT104, U302 and some other serotypes are still predominantly limited to S. Typhimurium strains.

Method of determining a geophysical-scale sea ice rheology from laboratory measurements

We present a methodology that allows a sea ice rheology, suitable for use in a General Circulation Model (GCM), to be determined from laboratory and tank experiments on sea ice when combined with a kinematic model of deformation. The laboratory experiments determine a material rheology for sea ice, and would investigate a nonlinear friction law of the form τ ∝ σ n⅔, instead of the more familiar Amonton's law, τ = μσn (τ is the shear stress, μ is the coefficient of friction and σ n is the normal stress). The modelling approach considers a representative region R containing ice floes (or floe aggregates), separated by flaws. The deformation of R is imposed and the motion of the floes determined using a kinematic model, which will be motivated from SAR observations. Deformation of the flaws is inferred from the floe motion and stress determined from the material rheology. The stress over R is then determined from the area-weighted contribution from flaws and floes

Bollène-2002 experiment: radar quantitative precipitation estimation in the Cévennes–Vivarais region, France

The Bollène-2002 Experiment was aimed at developing the use of a radar volume-scanning strategy for conducting radar rainfall estimations in the mountainous regions of France. A developmental radar processing system, called Traitements Régionalisés et Adaptatifs de Données Radar pour l’Hydrologie (Regionalized and Adaptive Radar Data Processing for Hydrological Applications), has been built and several algorithms were specifically produced as part of this project. These algorithms include 1) a clutter identification technique based on the pulse-to-pulse variability of reflectivity Z for noncoherent radar, 2) a coupled procedure for determining a rain partition between convective and widespread rainfall R and the associated normalized vertical profiles of reflectivity, and 3) a method for calculating reflectivity at ground level from reflectivities measured aloft. Several radar processing strategies, including nonadaptive, time-adaptive, and space–time-adaptive variants, have been implemented to assess the performance of these new algorithms. Reference rainfall data were derived from a careful analysis of rain gauge datasets furnished by the Cévennes–Vivarais Mediterranean Hydrometeorological Observatory. The assessment criteria for five intense and long-lasting Mediterranean rain events have proven that good quantitative precipitation estimates can be obtained from radar data alone within 100-km range by using well-sited, well-maintained radar systems and sophisticated, physically based data-processing systems. The basic requirements entail performing accurate electronic calibration and stability verification, determining the radar detection domain, achieving efficient clutter elimination, and capturing the vertical structure(s) of reflectivity for the target event. Radar performance was shown to depend on type of rainfall, with better results obtained with deep convective rain systems (Nash coefficients of roughly 0.90 for point radar–rain gauge comparisons at the event time step), as opposed to shallow convective and frontal rain systems (Nash coefficients in the 0.6–0.8 range). In comparison with time-adaptive strategies, the space–time-adaptive strategy yields a very significant reduction in the radar–rain gauge bias while the level of scatter remains basically unchanged. Because the Z–R relationships have not been optimized in this study, results are attributed to an improved processing of spatial variations in the vertical profile of reflectivity. The two main recommendations for future work consist of adapting the rain separation method for radar network operations and documenting Z–R relationships conditional on rainfall type.

EISCAT observations of plasma convection and the high-latitude, winter F-region during substorm activity

A 24 h period of observations by the EISCAT radar and other ground-based instrumentation is used to study the role of plasma convection in determining the morphology of the high-latitude F-region during winter. It is suggested that, in the afternoon sector of the polar convection pattern, rapid zonal (westward) flows caused low F-region electron densities due to an extension of the mid-latitude trough far into the sunlit hemisphere. Low densities on the dawn side prior to 0600 UT may also have been due to a trough-like feature. Although the generation mechanism is unclear, the trough may be the fossil remnant of a substorm. Around midnight, high F-region densities were seen, probably due to plasma flow emerging from the cap through soft particle precipitation in the auroral oval. Two substorms occurred at times when the radar was south of the auroral oval. Both caused enhanced convection speeds, a swing to equatorward flow, enhanced E-region densities and a depleted F-region. The first was seen as a Westward Travelling Surge, and the swing to purely southward flow which followed the surge front did not return to westward flows until 80–110 min later. The Harang discontinuity was observed co-rotating eastwards between the substorms, 65 ± 30 min before the separatrix between the dawn and dusk convection cells.