Persistent Staphylococcus aureus isolates from two independent cases of bacteremia display increased bacterial fitness and novel immune evasion phenotypes.

Staphylococcus aureus bacteremia cases are complicated by bacterial persistence and treatment failure despite the confirmed in vitro susceptibility of the infecting strain to administered antibiotics. A high incidence of methicillin-resistant S. aureus (MRSA) bacteremia cases are classified as persistent and are associated with poorer patient outcomes. It is still unclear how S. aureus evades the host immune system and resists antibiotic treatment for the prolonged duration of a persistent infection. In this study, the genetic changes and associated phenotypic traits specific to S. aureus persistent bacteremia were identified by comparing temporally dispersed isolates from persistent infections (persistent isolates) originating from two independent persistent S. aureus bacteremia cases with the initial infection isolates and with three resolved S. aureus bacteremia isolates from the same genetic background. Several novel traits were associated specifically with both independent sets of persistent S. aureus isolates compared to both the initial isolates and the isolates from resolved infections (resolved isolates). These traits included (i) increased growth under nutrient-poor conditions; (ii) increased tolerance of iron toxicity; (iii) higher expression of cell surface proteins involved in immune evasion and stress responses; and (iv) attenuated virulence in a Galleria mellonella larva infection model that was not associated with small-colony variation or metabolic dormancy such as had been seen previously. Whole-genome sequence analysis identified different single nucleotide mutations within the mprF genes of all the isolates with the adaptive persistence traits from both independent cases. Overall, our data indicate a novel role for MprF function during development of S. aureus persistence by increasing bacterial fitness and immune evasion.

Identifying the seasonal origins of human campylobacteriosis.

Human campylobacteriosis exhibits a distinctive seasonality in temperate regions. This paper aims to identify the origins of this seasonality. Clinical isolates [typed by multi-locus sequence typing (MLST)] and epidemiological data were collected from Scotland. Young rural children were found to have an increased burden of disease in the late spring due to strains of non-chicken origin (e.g. ruminant and wild bird strains from environmental sources). In contrast the adult population had an extended summer peak associated with chicken strains. Travel abroad and UK mainland travel were associated with up to 17% and 18% of cases, respectively. International strains were associated with chicken, had a higher diversity than indigenous strains and a different spectrum of MLST types representative of these countries. Integrating empirical epidemiology and molecular subtyping can successfully elucidate the seasonal components of human campylobacteriosis. The findings will enable public health officials to focus strategies to reduce the disease burden.

Campylobacter genotypes from poultry transportation crates indicate a source of contamination and transmission.

AIMS: Crates used to transport live poultry can be contaminated with Campylobacter, despite periodic sanitization, and are potential vectors for transmission between flocks. We investigated the microbial contamination of standard and silver ion containing crates in normal use and the genetic structure of associated Campylobacter populations. METHODS AND RESULTS: Bacteria from crates were enumerated by appropriate culture techniques, and multilocus sequence typing (MLST) was used to determine the genetic structure of Campylobacters isolated from standard and silver ion containing crates. Compared to standard crates, counts of bacteria, including Campylobacter, were consistently lower on silver ion containing crates throughout the decontamination process. In total, 16 different sequence types were identified from 89 Campylobacter jejuni isolates from crates. These were attributed to putative source population (chicken, cattle, sheep, the environment, wild bird) using the population genetic model, structure. Most (89%) were attributed to chicken, with 22% attribution to live chicken and 78% to retail poultry meat. MLST revealed a progressive shift in allele frequencies through the crate decontamination process. Campylobacter on crates survived for at least 3 h after sanitization, a period of time equivalent to the journey from the processing plant to the majority of farms in the catchment, showing the potential for involvement of crates in transmission. CONCLUSIONS: Inclusion of a silver ion biocide in poultry transportation crates to levels demonstrating acceptable antibacterial activity in vitro reduces the level of bacterial contamination during normal crate use compared to standard crates. Molecular analysis of Campylobacter isolates indicated a change in genetic structure of the population with respect to the poultry-processing plant sanitization practice. SIGNIFICANCE AND IMPACT OF THE STUDY: The application of a sustainable antimicrobial to components of poultry processing may contribute to reducing the levels of Campylobacter circulating in poultry.

Multi-locus sequence types of Campylobacter carried by flies and slugs acquired from local ruminant faeces.

AIMS: To assess whether flies and slugs acquire strains of Campylobacter jejuni and Campylobacter coli present in local ruminant faeces. METHODS AND RESULTS: Campylobacter was cultured from flies, slugs and ruminant faeces that were collected from a single farm in Scotland over a 19-week period. The isolates were typed using multi-locus sequence typing (MLST) and compared with isolates from cattle and sheep faeces. Campylobacter jejuni and Camp. coli were isolated from 5·8% (n=155, average of 75 flies per pool) and 13·3% (n=15, average of 8·5 slugs per pool) of pooled fly and slug samples, respectively. The most common sequence type (ST) in flies was Camp. coli ST-962 (approx. 40%) regardless of the prevalence in local cattle (2·3%) or sheep (25·0%) faeces. Two positive slug pools generated the same ST that has not been reported elsewhere. CONCLUSIONS: Despite their low carriage rate, flies are able to acquire Campylobacter STs that are locally present, although the subset carried may be biased when compared to local source. Slugs were shown to carry a previously unreported Campylobacter ST. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has demonstrated that flies carry viable Campylobacter and may contribute to the transfer of STs within and between groups of animals on farms. Further, they may therefore present a risk to human health via their contact with ready-to-eat foods or surfaces.

MLST clustering of Campylobacter jejuni isolates from patients with gastroenteritis, reactive arthritis and Guillain-Barré syndrome.

AIMS: To determine the diversity and population structure of Campylobacter jejuni (C. jejuni) isolates from Danish patients and to examine the association between multilocus sequence typing types and different clinical symptoms including gastroenteritis (GI), Guillain-Barré syndrome (GBS) and reactive arthritis (RA). METHODS AND RESULTS: Multilocus sequence typing (MLST) was used to characterize 122 isolates, including 18 from patients with RA and 8 from patients with GBS. The GI and RA isolates were collected in Denmark during 2002-2003 and the GBS isolates were obtained from other countries. In overall, 51 sequence types (STs) were identified within 18 clonal complexes (CCs). Of these three CCs, ST-21, ST-45 and ST-22 clonal complexes accounted for 64 percent of all isolates. The GBS isolates in this study significantly grouped into the ST-22 clonal complex, consistent with the PubMLST database isolates. There was no significant clustering of the RA isolates. CONCLUSIONS: Isolates from Denmark were found to be highly genetically diverse. GBS isolates grouped significantly with clonal complex ST-22, but the absence of clustering of RA isolates indicated that the phylogenetic background for this sequela could not be reconstructed using variation in MLST loci. Possibly, putative RA-associated genes may vary, by recombination or expression differences, independent of MLST loci. SIGNIFICANCE AND IMPACT OF THE STUDY: MLST typing of C. jejuni isolates from Danish patients with gastroenteritis confirmed that the diversity of clones in Denmark is comparable to that in other European countries. Furthermore, a verification of the grouping of GBS isolates compared to RA isolates provides information about evolution of the bacterial population resulting in this important sequela.

Evaluation of temperature gradient gel electrophoresis for the analysis of prey DNA within the guts of invertebrate predators.

The utility of temperature gradient gel electrophoresis (TGGE) as a means of analysing the gut contents of predators was evaluated. Generalist predators consume multiple prey species and a species-specific primer approach may not always be a practical means of analysing predator responses to prey diversity in complex and biodiverse ecosystems. General invertebrate primers were used to amplify the gut contents of predators, generating banding patterns that identified component prey remains. There was no evidence of dominance of the polymerase chain reaction (PCR) by predator DNA. When applied to field samples of the carabid predator Pterostichus melanarius (Illiger) nine banding patterns were detected, including one for aphids. To further distinguish between species, group-specific primers were designed to separate species of earthworm and aphid. TGGE of the earthworm PCR products generated banding patterns that varied with haplotype in some species. Aphid and earthworm DNA could be detected in the guts of carabids for up to 24 h using TGGE. In P. melanarius, with low numbers of prey per insect gut (mean<3), interpretation of banding patterns proved to be tractable. Potential problems of interpretation of TGGE gels caused by multiple prey bands, cryptic bands, haplotype variation, taxonomic uncertainties (especially with regard to earthworms), secondary predation, scavenging and presence of parasites and parasitoids in the prey or the predators, are discussed. The results suggest that PCR, using combinations of general invertebrate and group-specific primers followed by TGGE, provides a potentially useful approach to the analysis of multiple uncharacterized prey in predators.

Molecular detection of predation by soil micro-arthropods on nematodes.

The relative importance of the factors driving change in the population dynamics of nematodes in the soil is almost completely unknown. Top-down control by micro-arthropod predators may have a significant impact on nematode population dynamics. We report experiments showing that mites and Collembola were capable of reducing nematode numbers in the laboratory and were feeding on a targeted nematode species in the field. A PCR-based approach was developed for the detection of predation on three species of slug- and insect-pathogenic nematodes: Phasmarhabditis hermaphrodita, Heterorhabditis megidis and Steinernema feltiae. The collembolan Folsomia candida and the mesostigmatid mite Stratiolaelaps miles were employed as model predators to calibrate post-ingestion prey DNA detection times. Fragments of cytochrome oxidase I (COI) mtDNA were sequenced and species-specific primers were designed, amplifying 154-, 154- and 203-bp fragments for each of the nematode species. Detection times for nematode DNA within the guts of Collembola were longer than in mites, with half-lives (50% of samples testing positive) of 08.75 h and 05.03 h, respectively. F. candida significantly reduced numbers of the nematode H. megidis, with rates of predation of approximately 0.4 nematode infective juveniles per collembolan per hour over 10 h. Four taxa of field-caught micro-arthropod that had been exposed to the nematode P. hermaphrodita for a period of 12 h were analysed and significant numbers of three taxa tested positive. This is the first application of PCR techniques for the study of nematophagy and the first time these techniques have been used to measure predation on nematodes in the field.

Detection of secondary predation by PCR analyses of the gut contents of invertebrate generalist predators.

Predation by generalist predators is difficult to study in the field because of the complex effects of positive and negative interactions within and between predator species and guilds. Predation can be monitored by molecular means, through identification of prey DNA within predators. However, polymerase chain reaction (PCR) amplification of prey DNA from predators cannot discriminate between primary and secondary predation (hyperpredation), in which one predator feeds on another that has recently eaten the target prey. Here we quantify, for the first time, the potential error caused by detection of prey DNA following secondary predation, using an aphid-spider-carabid model. First, the aphid Sitobion avenae was fed to the spider Tenuiphantes tenuis and the carabid Pterostichus melanarius, and the postconsumption detection periods, for prey DNA within predators, were calculated. Aphids were then fed to spiders and the spiders to carabids. Aphid DNA was detected in the predators using primers that amplified 245- and 110-bp fragments of the mitochondrial cytochrome oxidase I gene. Fragment size and predator sex had no significant effect on detection periods. Secondary predation could be detected for up to 8 h, when carabids fed on spiders immediately after the latter had consumed aphids. Beetles tested positive up to 4 h after eating spiders that had digested their aphid prey for 4 h. Clearly, the extreme sensitivity of PCR makes detection of secondary predation more likely, and the only reliable answer in future may be to use PCR to identify, in parallel, instances of intraguild predation.

The impact of sludge amendment on gas dynamics in an upland soil: monitored by membrane inlet mass spectrometry.

Studies of the land disposal of biosolids and municipal sewage have focused largely on the potential pollution of the soil with pathogens, toxic compounds or heavy metals. Little is known about the impact of sludge amendment on carbon source and sink concentrations in soils. In this study gas concentrations in Scottish soil cores (from limed and unlimed plots) were monitored continuously at 3 cm depth before, during and after sludge application using membrane inlet mass spectrometry (MIMS). Following sludge application to soil cores, significant and sustained increases in CH4 (for 8 days) and CO2 (for between 16 and 120 days) concentration were observed. This suggested short-term stimulation of indigenous methanogens, provision of a new methanogenic inoculum, or inhibition of methane oxidizers (for example by heavy metals or NH4 in sludge). Soil microbial fermentative activity was enhanced over periods of a few months as shown by elevated CO2 concentrations.

Infiltration by alien predators into invertebrate food webs in Hawaii: a molecular approach.

Abstract Alien invertebrate predators have been introduced to Hawaii to control pests, particularly in lowland areas where most crops are grown. We developed techniques for assessing the impact of these predators on native food webs in relatively pristine upland areas where, it was hypothesized, few lowland predators might be found. Predator densities were assessed along transects within the Alakaii Swamp on Kaua'i. The most numerous alien biocontrol agents found were Halmus chalybeus (Coccinellidae), a species known to feed on Lepidoptera eggs. Laboratory experiments were conducted using two genera of endemic Lepidoptera, Scotorythra and Eupithecia (Geometridae), that are of considerable conservation value, the former because of its recent speciation across Hawaii, the latter for its unique predatory larvae. Techniques were developed for detecting Lepidoptera DNA within the guts of alien predators using prey-specific PCR primers. General primers amplified fragments of the mitochondrial cytochrome oxidase I gene from beetles and Lepidoptera. The sequences were aligned and used successfully to design target-specific primers for general detection of the remains of Geometridae and for particular species, including Scotorythra rara and Eupithecia monticolans. DNA fragments amplified were short [140-170 base pairs (bp)], optimizing detection periods following prey ingestion. Trials using the introduced biocontrol agent Curinus coeruleus (Coccinellidae) demonstrated detection of Lepidoptera DNA fragments = 151 bp in 85-100% of beetles after 24 h digestion of an early instar larva. This study provides a framework for future use of molecular gut analysis in arthropod conservation ecology and food web research with considerable potential for quantifying threats to endemic species in Hawaii and elsewhere.

Effects of soil amendment on gas depth profiles in soil monoliths using direct mass spectrometric measurement.

Land use and agricultural practices are known to influence the source and sink concentrations of various gases, including greenhouse gases (NOx CH4 and CO2). in soils. With everincreasing production of domestic sewage sludge and the prohibition of disposal at sea, pressure on waste disposal increases. Anaerobically digested domestic sewage sludge and/or lime were applied to an upland. Scottish soil and their effects on gas depth profiles monitored as indicators of microbial processes of the soil ecosystem. The concentrations of various gases (Ar, O2. CO2, CH4, N2, NOx) were measured simultaneously at each depth using membrane inlet mass spectrometry (MIMS). This technique enables the direct measurement of multiple gas species throughout soil cores with minimal disturbance. Intact soil monoliths were collected from the sample site, following amendment, and maintained in a constant temperature, environmental growth chambers. Statistical analyses (one-way ANOVA and LSD tests) were conducted to identify the depths at which gas concentrations in amended cores were significantly different from those in control (un-amended) cores. Significant effects were observed on the concentration of CO2, CH4, NOx and N2 at certain depths. Average CH4 concentration was consistently higher (>1 microM) in the upper horizon following application of sludge and sludge and lime together. N2 and NOx concentrations were elevated in cores treated with lime by approximately 100 and 32 microM. respectively, in much of the upper horizon. CO2 concentration increased above control mean values, at certain depths, following application of either sludge or lime. Some explanation for the changes in soil gas concentration was provided by reference to the microorganism assemblages and the gases associated with biochemistry of nitrification, denitrification, methane oxidation and methanogenesis.

Direct mass spectrometric measurement of gases in soil monoliths.

An integrated approach to gas analysis in soil cores was conducted to provide a novel method for observing the gas dynamics associated with upland soil ecosystems. Depth profiles of the O(2), Ar, CO(2), CH(4), N(2) and NO(x) concentrations in intact soil monoliths were obtained simultaneously using membrane inlet mass spectrometry (MIMS). This technique enables the direct measurement of multiple gas species throughout the soil core with minimal disturbance. Depth profiles provided data on the vertical heterogeneity of gas concentrations, while horizontal heterogeneity was monitored by comparison between profiles. Detailed descriptions of the modifications to current MIMS methods for in situ environmental monitoring of terrestrial soils are provided. These included a thorough examination of calibration of the MIMS probe in gas phase, stirred and unstirred H(2)O, or between glass beads immersed in H(2)O. Calibration was also carried out in sterile (autoclaved) soil. The mean concentrations of CO(2) and CH(4) in the soil monoliths increased from 27 microM and undetectable levels respectively at the surface, to maximum values of 3.6 mM and 4.3 microM at 12-cm depth. These changes corresponded with decreases in mean O(2), Ar and N(2) concentration from 300, 20 and 720 microM respectively to 0-6, 10 and 574 microM at 12-cm depth. These data indicated the presence of a gradient within the core from an aerobic environment to an O(2)-depleted, but not in all cases a completely anaerobic, one. This transition corresponded, to some extent, with that between the upper and lower soil horizons. The increased methane and CO(2) concentrations observed at depth are indicative of anaerobic environments. General trends associated with the gradually changing vertical heterogeneity of these gas profiles and the transition towards anaerobiosis did not provide evidence for the existence of localised microsites. Some evidence for microsite-specific microbial communities was however, provided by observation of broad zones of accumulation of NO(x) species, but only at concentrations close to the limit of detection of the method. The ratio of each gas, to argon was calculated at each depth. This was done to correct for physical parameters, which influence inert and biologically active gases, equally. The amount of di-nitrogen as a ratio to Ar was seen to increase with depth. This could be evidence for denitrification in the lower horizon. An example of the dynamic 'online' data collection capabilities is provided for diurnal oscillations in subsurface (5 cm) soil gas concentrations.

A high-resolution fast spin-echo inversion-recovery sequence for preoperative localization of the internal globus pallidus.

A fast spin-echo inversion-recovery (FSE-IR) sequence is described for its utility regarding surgical planning for patients with Parkinson's disease (PD) who are undergoing microelectrode-guided internal globus pallidus (GPi) ablation. Images from thirty-seven adult patients with PD were reviewed and visualization of the GPi, globus pallidus externa (GPe), and the intervening lamina was noted. High-resolution images were acquired from all patients despite the external hardware and the patients' movement disorder. In all cases, the conventional surgical trajectory, determined indirectly by a fixed measurement from the anteroposterior commissure line, was modified by the ability to visualize the GPi and optic tract directly. This sequence facilitated accurate stereotactic targeting.

Pre- and postoperative MR evaluation of stereotactic pallidotomy.

PURPOSE: Stereotactic pallidotomy, which has evolved as a result of technological advances in high-resolution MR imaging and microelectrode electrophysiological recording, is becoming a major form of treatment for patients with Parkinson disease in whom medical therapy has failed. We describe the location and appearance of the pallidotomy lesion on high-resolution MR images. METHODS: MR images in 83 patients (60 men and 23 women) who underwent stereotactic pallidotomy were reviewed retrospectively. The prepallidotomy screening study included standard spin-echo and gradient-echo sequences. After placement of a stereotactic headframe, volume-acquisition T1-weighted spoiled gradient-echo images were acquired for target localization in the posteroventral internal globus pallidus. One to three days after the pallidotomy, volume-acquisition T1-weighted and standard spin-echo sequences were obtained. In 16 patients, turbo spin-echo inversion recovery images also were obtained before and after surgery. The diameter, signal intensity, and location of the lesions relative to the midcommissural point and the intercommissural line were noted. RESULTS: The average lesion volume was 118 mm3 while that of the lesion-edema complex was 420 mm3. The midportion of the lesion was located on average 3.5 mm anterior to the midcommissural point, 21 mm lateral to the middle of the third ventricle, and 1.2 mm inferior to the intercommissural line. Signal intensity of the lesions varied, but all had a rim of edema. Forty-two patients had edema extending into the optic tract, four had increased signal in the ipsilateral basal ganglia on T2-weighted images, and seven had hemorrhage involving the ipsilateral caudate, internal capsule, and putamen. All patients experienced some improvement in contralateral bradykinesia, rigidity, and dystonia. CONCLUSION: The acute pallidotomy lesion is invariably located within the posteroventral internal globus pallidus, is usually hyperintense centrally on T1-weighted and turbo spin-echo inversion recovery MR images, and has a thin rim of edema. Edema extending into the ipsilateral optic tract was a common finding, but this series of patients evinced no visual changes.

T2-weighted three-dimensional turbo spin-echo MR of intracranial aneurysms.

A T2-weighted three-dimensional turbo spin-echo sequence is described that accurately depicted the anatomy of 18 intracranial aneurysms in 10 patients.

Incidence of aneurysm formation after Dacron patch aortoplasty repair for coarctation of the aorta: long-term results and assessment utilizing magnetic resonance angiography with three-dimensional surface rendering.

OBJECTIVES: Magnetic resonance angiography with three-dimensional surface rendering was performed to determine its value in assessing anatomic detail in patients with suspected aortic aneurysms. BACKGROUND: Dacron patch aortoplasty repair of coarctation of the aorta carries an inherent risk of aneurysm development. Sudden death from aortic rupture prompted discontinuing this operation and evaluating 39 patients (16 girls; mean age 6.3 years, range 10 days to 14.5 years) undergoing repair between January 1976 and October 1987. The aorta ruptured in 10 patients; 6 died at a mean interval of 8.1 years (range 0.75 to 12.4) after repair. All 33 survivors were interviewed and examined. METHODS: Conventional magnetic resonance imaging was performed in 26 patients, magnetic resonance angiography in 18. Angiographic slices were used to reconstruct three-dimensional images. No catheterization or contrast angiography was performed. Surgical intervention was based on clinical findings and magnetic resonance images. RESULTS: Twenty patients (11 girls) developed aneurysms, of which nine were detected in patients studied by magnetic resonance. Ruptures occurred in eight female patients, three of whom were pregnant. Surface renderings accurately defined aortic anatomy or aneurysms in all patients. On follow-up, no aneurysms have been detected in patients with negative magnetic resonance study results. Precise anatomic correlation with operative findings was reported. CONCLUSIONS: Magnetic resonance angiography with three-dimensional surface rendering provides noninvasive, radiation-free and contrast agent-free high resolution images of the thoracic aorta. These images can be reviewed and have three-dimensional form and perspective. These techniques were preferred over invasive angiography by surgeons and clinicians as definitive, risk-free procedures before surgical intervention.

Color map of contrast enhancement on MR images: use of desktop computers.

An alternative method of presenting and evaluating unenhanced and contrast-enhanced MR images is illustrated. We used a desktop computer and commercially available software to combine unenhanced and contrast-enhanced images to produce a color map of contrast enhancement superimposed on a gray-scale image. The color-enhanced images increased the conspicuousness of the areas of contrast enhancement, which is particularly important if the enhancing structures are small or adjacent to fatty tissues. The process has advantages over standard subtraction techniques in that gray scale is preserved while areas of subtle contrast enhancement are readily seen as areas of color. Although applied to MR imaging, this method may also be applicable to other studies that use contrast enhancement.