In-depth prospection of Avène Thermal Spring Water reveals an uncommon and stable microbial community.

BACKGROUND: Avène Thermal Spring Water (TSW) exhibits therapeutic properties in the treatment of skin pathologies. Arising from a dolomitic aquifer system, its physico-chemical properties are well-established and its bacteriological quality regularly monitored. The microbiota of this aquifer have been characterized. OBJECTIVES: We aimed to describe the structure of the bacterial community inhabiting the deep aquifer and to examine its dynamics over time. METHODS: The Avène TSW was collected at the catchment point and filtered through 0.1 µm pore size filters. The sampling was carried out every 3 months to generate a 4-year time series. The DNA extracted from filters was analysed using high-throughput 16S rRNA gene amplicon sequencing, and the microorganisms and their contribution were characterized by the taxonomic assignment of sequence variants generated from each sample. RESULTS: Bacteria were distributed into 39 phyla. Nitrospirae and Proteobacteria were the most prevalent, accounting for 38% and 23% of the total community on average, respectively. A stable pattern was observed throughout the study. A few bacterial species were always detected, forming a core community of likely chemolithoautotrophic organisms which might use energy sources and nutrients produced from water-bedrock interactions. Most of the species were distantly related to organisms described to date. CONCLUSIONS: Avène TSW provided by the deep aquifer system harbours a unique microbial community, shaped by the physico-chemical characteristics of the deep environment. Its remarkable stability over time has revealed a high level of confinement of the water resource.

Impact of seawater-quality and water treatment procedures on the active bacterial assemblages at two desalination sites.

Inorganic and organic compounds, particles and microorganisms in intake waters are mainly responsible for fouling of reverse osmosis membranes, which reduces the efficiency of the desalination process. The characterization of seawater quality to better predict its fouling potential remains a challenge for the desalination field and little is known about the seasonal variability of water quality parameters in the coastal waters used to supply desalination plants. In this study, standard water quality methods were combined with flow cytometry and molecular methods (16S rRNA sequencing and fingerprinting) to assess in parallel, the physicochemical properties, the microbial abundance and the active microbial community composition of the intake waters and their associated pretreated waters at two desalination sites from July 2007 to July 2008. The overall assessment of quality parameters revealed that microfiltration followed by slow sand filtration were the most efficient in removing microorganisms than the conventional dual media filtration routinely used in full-scale desalination plants, and that all treatments were inefficient for organic matter reduction. Temporal variation of the environmental parameters such as temperature, turbidity and silt density index only moderately affected the bacterial community structure in raw waters, but that interestingly, water treatment compartments changed the composition and diversity of the metabolically active bacterial populations and thus create distinct ecological post-treatment niches.

High diversity and abundance of Legionella spp. in a pristine river and impact of seasonal and anthropogenic effects.

The diversity and dynamics of Legionella species along a French river watershed subject to different thermal and wastewater discharges during an annual cycle were assessed by 16S rRNA gene sequencing and by a fingerprint technique, single-strand conformation polymorphism. A high diversity of Legionella spp. was observed at all the sampling sites, and the dominant Legionella clusters identified were most closely related to uncultured bacteria. The monthly monitoring revealed that Legionella sp. diversity changes were linked only to season at the wastewater site whereas there was some evidence for anthropogenic effects on Legionella sp. diversity downstream of the thermal bath. Quantification of Legionella pneumophila and Legionella spp. by culture and quantitative PCR (qPCR) was performed. Whereas only L. pneumophila was quantified on culture media, the qPCR assay revealed that Legionella spp. were ubiquitous and abundant from the pristine source of the river to the downstream sampling sites. These results suggest that Legionella spp. may be present at significant concentrations in many more freshwater environments than previously thought, highlighting the need for further ecological studies and culturing efforts.

Extramural vascular invasion is an adverse prognostic indicator of survival in patients with colorectal cancer.

BACKGROUND/AIMS: Extramural vascular invasion (EVI) in colorectal cancer is reported to be a stage-independent adverse prognostic factor, and is a core item in the Royal College of Pathologists minimum data set for colorectal cancer histopathology reporting. The detection of EVI is also highly variable amongst pathologists. Our aims were to analyse both the frequency of EVI in colorectal cancer resections, and the effect of EVI on survival, in patients operated on over a 5-year period. METHOD: A retrospective analysis of patients having potentially curative surgery for colorectal cancer between January 1999 and December 2004. RESULTS: Over 5 years, 378 patients underwent a potentially curative resection. One-hundred seven (28.3%) cancers exhibited EVI, of which 104 (97%) were T3 and T4 tumours. Survival curves with and without EVI, unadjusted for nodal status and T stage, were significantly different (P = 0.0001) with 5-year survivals of 52% and 73% respectively. Survival curves for T3 and T4 tumours stratified with and without EVI also showed significantly different survival distributions (P = 0.007). A significant difference in frequency of EVI year on year was seen (P < 0.001), ranging from 8.5% to 46.7%, whereas the number of T3 and T4 tumours in each year was not significantly different (P = 0.677). CONCLUSIONS: EVI is an adverse prognostic indicator for survival in patients undergoing potentially curative resection of colorectal cancer, and the routine requirement of EVI in colorectal cancer histopathology reporting is justified. Optimal specimen preparation, meticulous histopathological analysis and regular auditing of EVI detection rates are essential for the accurate staging of colorectal cancer.

The NHS Bowel Cancer Screening Programme--a realistic approach with additional benefits.

Colorectal cancer (CRC) is the third most common cancer and the second most common cause of cancer death in the UK with 36 100 new cases diagnosed each year in England and Wales and 55% of all patients presenting with lymph node metastases at the time of diagnosis. Early detection, before the development of symptoms, may be an effective way of reducing mortality and it is this which a screening programme seeks to address. The NHS Bowel Cancer Screening Programme (NHS BCSP) commenced in April 2006 and invites men and women aged 60-69 to participate via submission of a faecal occult blood test every 2 years; those with a positive result will be offered colonoscopy as the next investigation of choice. This article will explore the background to the programme, including the financial considerations behind it and the implication that this has had on colonoscopy standards and training in the UK. The chosen programme is not the most effective neither in terms of survival benefit nor cost effectiveness but is a compromise within a financially strained health care system. Endoscopy standards because of its introduction have, however, considerably improved in terms of patient experience, safety and improved practice.

Rapid quantification of the toxic alga Prymnesium parvum in natural samples by use of a specific monoclonal antibody and solid-phase cytometry.

The increasing incidence of harmful algal blooms around the world and their associated health and economic effects require the development of methods to rapidly and accurately detect and enumerate the target species. Here we describe use of a solid-phase cytometer to detect and enumerate the toxic alga Prymnesium parvum in natural samples, using a specific monoclonal antibody and indirect immunofluorescence. The immunoglobulin G antibody 16E4 exhibited narrow specificity in that it recognized several P. parvum strains and a Prymnesium nemamethecum strain but it did not cross-react with P. parvum strains from Scandinavia or any other algal strains, including species of the closely related genus Chrysochromulina. Prymnesium sp. cells labeled with 16E4 were readily detected by the solid-phase cytometer because of the large fluorescence signal and the signal/noise ratio. Immunofluorescence detection and enumeration of cultured P. parvum cells preserved with different fixatives showed that the highest cell counts were obtained when cells were fixed with either glutaraldehyde or formaldehyde plus the cell protectant Pluronic F-68, whereas the use of formaldehyde alone resulted in significantly lower counts. Immunofluorescence labeling and analysis with the solid-phase cytometer of fixed natural samples from a bloom of P. parvum occurring in Lake Colorado in Texas gave cell counts that were close to those obtained by the traditional method of counting using light microscopy. These results show that a solid-phase cytometer can be used to rapidly enumerate natural P. parvum cells and that it could be used to detect other toxic algae, with an appropriate antibody or DNA probe.

Haemangiopericytoma of the sigmoid mesentery.

Haemangiopericytoma is a rare, vascular soft tissue tumour originating from the pericytes surrounding capillaries. We report a case of haemangiopericytoma in the sigmoid mesocolon and are aware of only one previously case. A 61-year-old man was referred with a left iliac fossa mass. At operation, a 10-cm diameter mass was found to be arising from the sigmoid mesentery (Fig. 1). The mass did not involve the bowel wall and there was no clinical evidence of metastatic disease. A sigmoid colectomy with primary anastomosis was performed. The patient made an uneventful recovery. Pathological assessment of the specimen revealed a 95 x 70 x 50 mm(3), purple, lobulated mass within the sigmoid mesocolon adjacent to the bowel. Immunohistological analysis (positive CD34, focal factor VIII) was consistent with a diagnosis of a haemangiopericytoma. Complete excision with adequate margins remains the treatment of choice. We therefore suggest that patients be carefully followed for long periods and advised of the risk of long-term relapse.

Lessons to be learned: a case study approach. Chronic intussusception in childhood.

Chronic intussusception as a cause of persistent abdominal pain in children is often an overlooked diagnosis. Here we present an eight-year-old boy, who at the age of three years had an acute intussusception reduced hydrostatically with barium and who subsequently had been extensively investigated both in Wales and in Switzerland, for persistent colicky abdominal pain. He was found to have chronic intussusception, with a Meckel's diverticulum being the cause of his symptoms.

Pyrolysis mass spectrometry analysis of free-living and symbiotic cyanobacteria.

The potential of pyrolysis mass spectrometry to distinguish closely related cyanobacterial strains was assessed by using the technique to compare symbiotic cyanobacteria isolated from the hornwort Phaeoceros laevis and free-living cyanobacterial strains at the same field site. The same strains had previously been compared using polymerase chain reaction-based DNA fingerprinting techniques (West & Adams 1997, Appl. Environ. Microbiol. 63: 4479-4484). Many of the strains were grouped identically by the two techniques, although there were some differences, possibly resulting from the ability of these cyanobacteria to develop a range of specialised cell types having different chemical compositions to the vegetative cells. Although growth conditions were chosen to suppress cellular differentiation, this may not always have been completely successful. With careful control of growth conditions pyrolysis mass spectrometry has considerable potential as an additional tool for the phenetic comparison of cyanobacterial strains. It has the advantage that analysis is directly derived from whole cells, and hence is simpler and cheaper than DNA-based methods, although it does require the growth of axenic strains. The technique may be particularly useful in the study of some of the more cryptic unicellular and non-heterocystous filamentous cyanobacterial groups, in which the lack of cellular differentiation should minimise any variability in the chemical composition of cells.

Niche-partitioning of Prochlorococcus populations in a stratified water column in the eastern North Atlantic Ocean.

The in situ community structure of Prochlorococcus populations in the eastern North Atlantic Ocean was examined by analysis of Prochlorococcus 16S rDNA sequences with three independent approaches: cloning and sequencing, hybridization to specific oligonucleotide probes, and denaturing gradient gel electrophoresis (DGGE). The hybridization of high-light (HL) and low-light (LL) Prochlorococcus genotype-specific probes to two depth profiles of PCR-amplified 16S rDNA sequences revealed that in these two stratified water columns, an obvious niche-partitioning of Prochlorococcus genotypes occurred. In each water column a shift from the HL to the LL genotype was observed, a transition correlating with the depth of the surface mixed layer (SML). Only the HL genotype was found in the SML in each water column, whereas the LL genotype was distributed below the SML. The range of in situ irradiance to which each genotype was subjected within these distinct niches was consistent with growth irradiance studies of cultured HL- and LL-adapted Prochlorococcus strains. DGGE analysis and the sequencing of Prochlorococcus 16S rDNA clones were in full agreement with the genotype-specific oligonucleotide probe hybridization data. These observations of a partitioning of Prochlorococcus genotypes in a stratified water column provide a genetic basis for the dim and bright Prochlorococcus populations observed in flow cytometric signatures in several oceanic provinces.

Side-chains in native and random coil protein conformations. Analysis of NMR coupling constants and chi1 torsion angle preferences.

The behaviour of amino acid side-chains in proteins in solution has been characterised by analysing NMR 3JHalphaH beta coupling constants and crystallographic chi1 torsion angles. Side-chains both in the core of native folded proteins and in situations where there is an absence of close packing including the random coil state have been considered. An analysis of experimental 3JHalphaH beta coupling constant data for ten proteins shows that in the core of native proteins a very close similarity is observed between the chi1 conformations adopted in solution and in crystals. There is clear evidence, however, for significant motional averaging about the chi1 torsion angles in solution. Using a model of a Gaussian distribution about the average torsion angles the extent of these fluctuations has been quantified; the standard deviation for the motion is 26 degrees, the fluctuations about chi1 in the protein core being similar in size to those found for main-chain phi torsion angles in solution. From the distribution of chi1 torsion angles in a data base of protein crystal structures, torsion angle populations and coupling constants have been predicted for a random coil polypeptide. Significant variations in the chi1 distributions for different amino acids give differences in the predicted coupling constants; for 3JHalphaH beta, for example, values of 5.1 and 5.7 Hz are predicted for serine compared with 4.9 and 9.9 Hz for leucine. Experimental data for short unstructured peptides show an excellent agreement with the predictions, indicating that the overall chi1 distributions in protein crystals reflect the local preferences of the amino acids. Predictions from the protein data base therefore provide an important framework for interpreting experimental data for non-native protein conformations and for residues on the surface of folded proteins.

Gentamicin and tobramycin pharmacokinetics in pediatric bone marrow transplant patients.

OBJECTIVE: To describe the pharmacokinetic parameters of gentamicin and tobramycin in pediatric bone marrow transplant patients. DESIGN: Retrospective medical record review. SETTING: Pediatric bone marrow transplant unit in a university teaching hospital. MAIN OUTCOME MEASURES: Pharmacokinetic parameters (apparent volume of distribution [Vd] in L/kg, half-life [t1/2] in h, elimination rate constant [ke] in h-1, clearance [Cl] in mL/min/1.73 m2 and mL/min/kg) calculated from serum concentrations. PATIENTS: Thirty-three patients aged 15 years or less who underwent bone marrow transplant and received gentamicin or tobramycin. RESULTS: Mean pharmacokinetic parameters were Vd 0.32 +/- 0.07 L/kg, t1/2 2.32 +/- 0.65 h, Cl 1.71 +/- 0.53 mL/min/kg, and Cl 86.2 +/- 24.5 mL/min/1.73 m2. Factors such as disease state, type of marrow graft, gender, or exposure to cyclosporine had no significant effect on pharmacokinetic parameters. Linear regression indicated a weak relationship between serum creatinine (SCr) and Cl in mL/min/kg (r = 0.59), but no relationship was found between SCr and Cl in mL/min/1.73 m2, between age and apparent Vd, or between SCr and apparent Vd. Models for estimating Cl and Ke developed by multiple regression were somewhat predictive (r = 0.7). Required calculated maintenance dosages to obtain therapeutic concentrations were 8, 7, and 6 mg/kg/d in children 6 or younger, 7-12, and 13-15 years, respectively. CONCLUSIONS: The mean Cl and apparent Vd for all ages are similar to those reported in pediatric oncology patients who had not undergone marrow transplantation. Children 6 years or younger had lower than expected Cls and larger apparent Vds than did the older children. Dosages estimated to be necessary to achieve therapeutic concentrations were 6-8 mg/kg/d.

Prevention and treatment of hemorrhagic cystitis.

Hemorrhagic cystitis is a syndrome associated with certain disease states as well as exposure to drugs, viruses, and toxins. It manifests as diffuse bleeding of the endothelial lining of the bladder. Treatment includes intravesical, systemic, and nonpharmacologic therapies, all of which have advantages and disadvantages.

Stability of tacrolimus in an extemporaneously compounded oral liquid.

The stability of tacrolimus in an extemporaneously compounded oral liquid formulation was studied. A suspension was prepared by mixing the contents of commercially available 5-mg capsules of tacrolimus with equal amounts of Ora-Plus and Simple Syrup, NF, to make a final volume of 60 mL. The final concentration of tacrolimus in the suspension was 0.5 mg/mL. Six identical suspensions were prepared, placed in three glass and three plastic amber prescription bottles, and stored at room temperature (24-26 degrees C). Immediately after preparation and at 7, 15, 30, 45, and 56 days, samples were removed and assayed in duplicate by stability-indicating high-performance liquid chromatography. At least 98% of the initial tacrolimus concentrations remained in all suspensions throughout the study period. Color, order, and pH did not change appreciably over the study period. Tacrolimus 0.5 mg/mL compounded extemporaneously in equal amounts of Ora-Plus and Simple Syrup, NF, was stable at 24-26 degrees C for at least 56 days in both glass and plastic amber prescription bottles.

Phenotypic and genotypic comparison of symbiotic and free-living cyanobacteria from a single field site.

PCR amplification techniques were used to compare cyanobacterial symbionts from a cyanobacterium-bryophyte symbiosis and free-living cyanobacteria from the same field site. Thirty-one symbiotic cyanobacteria were isolated from the hornwort Phaeoceros sp. at several closely spaced locations, and 40 free-living cyanobacteria were isolated from the immediate vicinity of the same plants. One of the symbiotic isolates was a species of Calothrix, a genus not previously known to form bryophyte symbioses, and the remainder were Nostoc spp. Of the free-living strains, two were Calothrix spp., three were Chlorogloeopsis spp. and the rest were Nostoc spp. All of the symbiotic and all but one of the free-living strains were able to reconstitute the symbiosis with axenic cultures of both Phaeoceros and the liverwort Blasia sp. Axenic cyanobacterial strains were compared by DNA amplification using PCR with either short arbitrary primers or primers specific for the regions flanking the 16S-23S rRNA internal transcribed spacer. With one exception, the two techniques produced complementary results and confirmed for the first time that a diversity of symbiotic cyanobacteria infect Phaeoceros in the field. Symbionts from adjacent colonies were different as often as they were the same, showing that the same thallus could be infected with many different cyanobacterial strains. Strains found to be identical by the techniques employed here were often found as symbionts in different thalli at the same locale but were never found free-living. Only one of the free-living strains, and none of the symbiotic strains, was found at more than one sample site, implying a highly localized distribution of strains.

Sterility of filgrastim (G-CSF) in syringes.

OBJECTIVE: To determine if the sterility of filgrastim (G-CSF) is maintained for up to 7 days when aseptically transferred from the vial to tuberculin syringes in a laminar air flow environment. DESIGN: The study was conducted in two phases: a validation and an experimental phase. The method was validated by inoculating samples of sterile filgrastim solution with common bacterial and fungal skin contaminants. Samples were aseptically drawn into syringes in a class 100 horizontal laminar air flow hood and refrigerated. The samples were equally divided and transferred to microbiology culture media at times 0, 24 hours, 48 hours, and 7 days; incubated; and the organisms identified and quantitated. In the experimental phase, samples of filgrastim were aseptically drawn into syringes, separated into three groups, and refrigerated. At 24 hours, 48 hours, and 7 days, the samples were transferred to broth, incubated, and observed for the development of turbidity. SETTING: A class 100 laminar air flow hood in a pediatric hospital pharmacy and a home-infusion pharmacy class 100,000 clean room. MAIN OUTCOME MEASURES: The sterility of filgrastim in syringes was determined by comparing experimental broth culture tubes to a control tube and observing for the development of turbidity. RESULTS: Filgrastim demonstrated the ability to support the growth of intentionally inoculated skin contaminants, both qualitatively and quantitatively. However, when aseptically transferred to syringes and refrigerated, all tested filgrastim samples remained sterile for at least 7 days. CONCLUSIONS: Syringes of filgrastim remain sterile for 7 days when prepared in a class 100 laminar air flow hood, using aseptic technique, and stored under refrigeration. This change in practice can result in significant cost savings.

Systemic antimicrobial treatment of foot infections in diabetic patients.

The pathophysiology, microbiology, and treatment of diabetic foot infections are reviewed. Patients with diabetes mellitus are at risk for developing infections of the lower extremity because of physiological and anatomical changes. The treatment must be aggressive to prevent systemic complications and recurrence. A combination of pathogens is likely to be found at the site of infection, including gram-negative and gram-positive aerobes as well as anaerobes. If preventive and nonpharmacologic treatment methods are not successful, systemic antimicrobial therapy is indicated. The appropriate agent for empirical therapy is chosen on the basis of the patient's medical history and clinical status with consideration to cost and administration issues. Until a specific organism is identified, a single broad-spectrum agent is administered. The duration of i.v. therapy and appropriate role for oral administration is based upon clinical response. Home infusion therapy is an option for medically stable patients. A single, broad-spectrum, i.v. antimicrobial is usually the best choice for empirical treatment of diabetic foot infection. The regimen is then tailored on the basis of the clinical response and culture and susceptibility test results. Aggressive pharmacologic and nonpharmacologic treatment is needed to avoid possible gangrene and loss of limb.