High-resolution characterization of short-term temporal variability in the taxonomic and resistome composition of wastewater influent.

Wastewater-based epidemiology (WBE) for population-level surveillance of antimicrobial resistance (AMR) is gaining significant traction, but the impact of wastewater sampling methods on results is unclear. In this study, we characterized taxonomic and resistome differences between single-timepoint-grab and 24 h composites of wastewater influent from a large UK-based wastewater treatment work [WWTW (population equivalent: 223 435)]. We autosampled hourly influent grab samples (n=72) over three consecutive weekdays, and prepared additional 24 h composites (n=3) from respective grabs. For taxonomic profiling, metagenomic DNA was extracted from all samples and 16S rRNA gene sequencing was performed. One composite and six grabs from day 1 underwent metagenomic sequencing for metagenomic dissimilarity estimation and resistome profiling. Taxonomic abundances of phyla varied significantly across hourly grab samples but followed a repeating diurnal pattern for all 3 days. Hierarchical clustering grouped grab samples into four time periods dissimilar in both 16S rRNA gene-based profiles and metagenomic distances. 24H-composites resembled mean daily phyla abundances and showed low variability of taxonomic profiles. Of the 122 AMR gene families (AGFs) identified across all day 1 samples, single grab samples identified a median of six (IQR: 5-8) AGFs not seen in the composite. However, 36/36 of these hits were at lateral coverage <0.5 (median: 0.19; interquartile range: 0.16-0.22) and potential false positives. Conversely, the 24H-composite identified three AGFs not seen in any grab with higher lateral coverage (0.82; 0.55-0.84). Additionally, several clinically significant human AGFs (bla VIM, bla IMP, bla KPC) were intermittently or completely missed by grab sampling but captured by the 24 h composite. Wastewater influent undergoes significant taxonomic and resistome changes on short timescales potentially affecting interpretation of results based on sampling strategy. Grab samples are more convenient and potentially capture low-prevalence/transient targets but are less comprehensive and temporally variable. Therefore, we recommend 24H-composite sampling where feasible. Further validation and optimization of WBE methods is vital for its development into a robust AMR surveillance approach.

Disposable nappies for preventing napkin dermatitis in infants.

BACKGROUND: Napkin dermatitis is a common condition that occurs in otherwise healthy infants. It causes discomfort to infants, anxiety to parents and caregivers and contributes to the load on the health care system. A large variety of napkins, both disposable and non-disposable, are available. Evidence is required to assist carers and health care workers in making informed decisions when balancing the pros and cons of different napkin choices. OBJECTIVES: To assess whether disposable napkins prevent napkin dermatitis in infants. SEARCH STRATEGY: We searched the Skin Group Specialised Register (up to June 2003), the Cochrane Central Register of Controlled Trials in (The Cochrane Library, Issue 3, 2004), MEDLINE (from 1966 to November 2004), EMBASE (from 1980 to February 2003) and CINAHL (from 1982 to November 2004). We searched reference lists of articles. We contacted lead investigators in the area and companies that manufacture disposable napkins for access to unpublished trials. SELECTION CRITERIA: Randomised controlled trials in which disposable napkins were compared with other types of disposable napkins or non-disposable napkins, in infants up to two years of age, for preventing napkin dermatitis. DATA COLLECTION AND ANALYSIS: Two authors independently extracted data. The same two authors independently assessed trials for methodological quality. Attempts were made to contact trial authors of the trials identified for clarification of methods and results of published trials. MAIN RESULTS: We identified 28 studies of the effects of various napkin types on napkin dermatitis. Seventeen studies from nine reports were included. Eleven studies were excluded due to methodology that did not fit the inclusion criteria of this review. Due to the poor reporting of methodology and results of the studies found in this review, there were no quantitative data available for analysis (or meta-analysis). Although the included studies appeared to favour cellulose-core disposable napkins over cloth, absorbent gelling material over cellulose-only core napkins, breathable outer shell over occlusive outer shell napkins and linings impregnated with formulations over plain linings, all of these studies were open to bias due to flawed methodology. AUTHORS' CONCLUSIONS: There is not enough evidence from good quality randomised controlled trials to support or refute the use and type of disposable napkins for the prevention of napkin dermatitis in infants.

Tubular spinae are long-distance connectors between bacteria.

The marine pseudomonad D71 (NCMB 2018) ['Spinomonas maritima'] can be induced to produce long tubular surface appendages (spinae) in a growth medium of low osmolarity. In general, spina-carrying cells show these appendages with open distal ends. We examined cultured cells by scanning and transmission electron microscopy, using both critical-point drying and thin sectioning after embedding with agarose protection. By scanning electron microscopy, spinae were observed that connected cells over distances of several micrometers. Ultrathin sections often revealed an additional layer outside the outer membrane, resembling an S-layer. The inner and outer cell membranes were often joined at spina-insertion areas. Furthermore, evidence was found in ultrathin sections for uninterrupted tubes connecting two cells over a distance of up to 7 microns. We propose, therefore, that spinae form the framework for wide open cell clusters; we hypothesize that these spinae might also permit an exchange of cell-to-cell signals.

Two bacteriophages of Clostridium difficile.

Two temperate bacteriophages of differing morphology and host range were isolated by screening 94 isolates of Clostridium difficile. Phage 41 had a 300-nm flexible tail, whereas phage 56 had a shorter tail with a contractile sheath. Electron microscopy of phage 56 lysates exposed to elevated magnesium concentrations showed small virus-like particles which were 21 nm in diameter. The addition of MgCl2 to semisolid agar overlays enhanced both the titer and plaque size of phage 56. Phage 56 was more temperature labile than phage 41 and demonstrated unusual lability in buffer at pH 7.0. One-step growth and adsorption experiments revealed that both phages had latent periods of about 60 min, but phage 56 adsorbed to its indicator strain more efficiently. Phage 56, which was obtained from a toxigenic strain of C. difficile, was used to lysogenize its nontoxigenic indicator strain, but no conversion to toxigenicity was observed in this strain.

Physicochemical and immunological homogeneity of spinin, the subunit-protein of bacterial spinae.

Bacterial spinae from marine bacterium D71 are multi-subunit structures of a single protein. This protein, called spinin, is homogeneous by immunodiffusion and immunoelectrophoresis, amino acid composition, polyacrylamide gel electrophoresis with a number of buffer systems, sedimentation velocity and diffusion boundary analysis. Sedimentation equilibrium gives Mr = 19,000, while phosphate polyacryl-amide gel electrophoresis in presence of dodecyl sulfate gives Mr = 32,000. The lower Mr estimate for spinin is supported by sedimentation equilibrium in 6 M guanidine . HCl, and covalent cross-linking with dimethyl suberimidate or glutaraldehyde. The higher Mr value probably arises from an anomalous spinin-dodecyl sulfate interaction. Isoelectric focusing in polyacrylamide gel gives pI = 3.45; however, the focusing pattern also contains three distinct bands that may arise from hydrolysis of the spinin protomer during anodic migration. This study presents the first extensive physicochemical characterization of spinin and provides the basis for investigating the subunit assembly of spinae.

Susceptibility of human embryonic kidneys in organ culture to herpesvirus hominis.

101 pairs of human embryonic kidneys of 5-12 weeks' gestation were maintained in whole organ culture by our previously described technique, with herpesvirus hominis types1 and 2 added to our regular media. One kidney of each pair served as a control and was exposed to identical culture medium without virus. Cultures were maintained for 24-120 h to study the time sequence of viral infectivity. Organs were then examined for the presence of virus by electron microscopy and histochemical staining. Histological studies of virus-infected kidneys showed either (1) complete organ death or (2) virus localization in undifferentiated cells, plus disorganization of architecture in differentiated areas. Control organs showed normal organization.

The attachment of bacterial spinae.

Spinae are attached to protease-sensitive structural proteins in the external surface of the outer membrane. Agents and (or) treatments affecting ionic, hydrophobic, or hydorgen bonds are ineffective in releasing spinae from bacteria. As judged by thin-sectioning and freeze-fracturing techniques, the outer membrane is not modified at the attachment site to a detectable extent, and the other surface layers are not involved. The attachment of spinae is thus differentiated from that of flagella.

Arrangement of morphological subunits in bacterial spinae.

The filament, that is helically arranged to form the bacterial spina, is composed of morphological subunits (oligomers) about 5.6 nm in width and 11 nm in length. The oligomers are asymmetrical in that the inner surface is grooved. Image analysis of negative-stained spinae ribbons indicates that the oligomers are paired, possibly beaded structures, the arrangement of which is easily distorted during preparation. In intact spinae, the oligomer orientation may be normal to the filament axis, but in collapsed freeze-etched spinae, the oligomers are inclined at a constant angle of about 72 degrees to the filament axis.

Ferritin in the fungus Phycomyces.

The iron-protein ferritin has been purified from mycelium, sporangiophores, and spores of the fungus Phycomyces blakesleeanus. It has a protein-to-iron ratio of 5, a sedimentation coefficient of 55S, a buoyant density in CsCl of 1.82 g/cm(3), and the characteristic morphology of ferritin in the electron microscope. Apoferritin prepared from Phycomyces ferritin has a sedimentation coefficient of 18S and consists of subunits of molecular weight 25,000. In the cytoplasm of Phycomyces, ferritin is located on the surface of lipid droplets (0.5-2.0 micro in diameter) where it forms crystalline monolayers which are conspicuous in electron micrographs of sporangiophore thin-sections. Ferritin is found in all developmental stages of Phycomyces but is concentrated in spores. The level of ferritin iron is regulated by the iron level in the growth medium, a 50-fold increase occurring on iron-supplemented medium.