Elucidation of the O-antigen structure of Escherichia coli O93 and characterization of its biosynthetic genes.

The structure of the O-antigen from the international reference strain Escherichia coli O93:-:H16 has been determined. A nonrandom modal chain-length distribution was observed for the lipopolysaccharide, a pattern which is typical when long O-specific polysaccharides are expressed. By a combination of (i) bioinformatics information on the gene cluster related to O-antigen synthesis including putative function on glycosyl transferases, (ii) the magnitude of NMR coupling constants of anomeric protons, and (iii) unassigned 2D 1H, 13C-HSQC, and 1H,1H-TOCSY NMR spectra it was possible to efficiently elucidate the structure of the carbohydrate polymer in an automated fashion using the computer program CASPER. The polysaccharide also carries O-acetyl groups and their locations were determined by 2D NMR experiments showing that ~½ of the population was 2,6-di-O-acetylated, ~» was 2-O-acetylated, whereas ~» did not carry O-acetyl group(s) in the 3-O-substituted mannosyl residue of the repeating unit. The structure of the tetrasaccharide repeating unit of the O-antigen is given by: →2)-ß-d-Manp-(1→3)-ß-d-Manp2Ac6Ac-(1→4)-ß-d-GlcpA-(1→3)-α-d-GlcpNAc-(1→, which should also be the biological repeating unit and it shares structural elements with capsular polysaccharides from E. coli K84 and K50. The structure of the acidic O-specific polysaccharide from Cellulophaga baltica strain NN015840T differs to that of the O-antigen from E. coli O93 by lacking the O-acetyl group at O6 of the O-acetylated mannosyl residue.

Structural elucidation of the O-antigen polysaccharide from Escherichia coli O125ac and biosynthetic aspects thereof.

Enteropathogenic Escherichia coli O125, the cause of infectious diarrheal disease, is comprised of two serogroups, viz., O125ab and O125ac, which display the aggregative adherence pattern with epithelial cells. Herein, the structure of the O-antigen polysaccharide from E. coli O125ac:H6 has been elucidated. Sugar analysis revealed the presence of fucose, mannose, galactose and N-acetyl-galactosamine as major components. Unassigned 1H and 13C NMR data from one- and two-dimensional NMR experiments of the O125ac O-antigen in conjunction with sugar components were used as input to the CASPER program, which can determine polysaccharide structure in a fully automated way, and resulted in the following branched pentasaccharide structure of the repeating unit: →4)[ß-d-Galp-(1 â†’ 3)]-ß-d-GalpNAc-(1 â†’ 2)-α-d-Manp-(1 â†’ 3)-α-l-Fucp-(1 â†’ 3)-α-d-GalpNAc-(1→, where the side chain is denoted by square brackets. The proposed O-antigen structure was confirmed by 1H and 13C NMR chemical shift assignments and determination of interresidue connectivities. Based on this structure, that of the O125ab O-antigen, which consists of hexasaccharide repeating units with an additional glucosyl group, was possible to establish in a semi-automated fashion by CASPER. The putative existence of gnu and gne in the gene clusters of the O125 serogroups is manifested by N-acetyl-d-galactosamine residues as the initial sugar residue of the biological repeating unit as well as within the repeating unit. The close similarity between O-antigen structures is consistent with the presence of two subgroups in the E. coli O125 serogroup.

Detection and isolation of airborne SARS-CoV-2 in a hospital setting.

Transmission mechanisms for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are incompletely understood. In particular, aerosol transmission remains unclear, with viral detection in air and demonstration of its infection potential being actively investigated. To this end, we employed a novel electrostatic collector to sample air from rooms occupied by COVID-19 patients in a major Swedish hospital. Electrostatic air sampling in conjunction with extraction-free, reverse-transcriptase polymerase chain reaction (hid-RT-PCR) enabled detection of SARS-CoV-2 in air from patient rooms (9/22; 41%) and adjoining anterooms (10/22; 45%). Detection with hid-RT-PCR was concomitant with viral RNA presence on the surface of exhaust ventilation channels in patients and anterooms more than 2 m from the COVID-19 patient. Importantly, it was possible to detect active SARS-CoV-2 particles from room air, with a total of 496 plaque-forming units (PFUs) being isolated, establishing the presence of infectious, airborne SARS-CoV-2 in rooms occupied by COVID-19 patients. Our results support circulation of SARS-CoV-2 via aerosols and urge the revision of existing infection control frameworks to include airborne transmission.

Evaluation of Bacteria and Fungi DNA Abundance in Human Tissues.

Whereas targeted and shotgun sequencing approaches are both powerful in allowing the study of tissue-associated microbiota, the human: microorganism abundance ratios in tissues of interest will ultimately determine the most suitable sequencing approach. In addition, it is possible that the knowledge of the relative abundance of bacteria and fungi during a treatment course or in pathological conditions can be relevant in many medical conditions. Here, we present a qPCR-targeted approach to determine the absolute and relative amounts of bacteria and fungi and demonstrate their relative DNA abundance in nine different human tissue types for a total of 87 samples. In these tissues, fungi genomes are more abundant in stool and skin samples but have much lower levels in other tissues. Bacteria genomes prevail in stool, skin, oral swabs, saliva, and gastric fluids. These findings were confirmed by shotgun sequencing for stool and gastric fluids. This approach may contribute to a more comprehensive view of the human microbiota in targeted studies for assessing the abundance levels of microorganisms during disease treatment/progression and to indicate the most informative methods for studying microbial composition (shotgun versus targeted sequencing) for various samples types.

Structural analysis of the O-antigen polysaccharide from Escherichia coli O188.

The structure of the O-antigen from Escherichia coli reference strain O188 (E. coli O188:H10) has been investigated. The lipopolysaccharide shows a typical nonrandom modal chain-length distribution and the sugar and absolute configuration analysis revealed d-Man, d-Glc, d-GlcN and d-GlcA as major components. The structure of the O-specific polysaccharide was determined using one- and two-dimensional 1H and 13C NMR spectroscopy experiments, where inter-residue correlations were identified by 1H,13C-heteronuclear multiple-bond correlation and 1H,1H-NOESY experiments, which revealed that it consists of pentasaccharide repeating units with the following structure: Biosynthetic aspects and NMR analysis are consistent with the presented structure as the biological repeating unit. The O-antigen of Shigella boydii type 16 differs only in that it carries O-acetyl groups to ~50% at O6 of the branch-point mannose residues. A molecular model of the E. coli O188 O-antigen containing 20 repeating units extends ~100 Å, which is similar to the height of the periplasmic portion of polysaccharide co-polymerase Wzz proteins that regulate the O-antigen chain length of lipopolysaccharides in the Wzx/Wzy biosynthetic pathway.

A fieldable electrostatic air sampler enabling tuberculosis detection in bioaerosols.

Tuberculosis (TB) infects about 25% of the world population and claims more human lives than any other infectious disease. TB is spread by inhalation of aerosols containing viable Mycobacterium tuberculosis expectorated or exhaled by patients with active pulmonary disease. Air-sampling technology could play an important role in TB control by enabling the detection of airborne M. tuberculosis, but tools that are easy to use and scalable in TB hotspots are lacking. We developed an electrostatic air sampler termed the TB Hotspot DetectOR (THOR) and investigated its performance in laboratory aerosol experiments and in a prison hotspot of TB transmission. We show that THOR collects aerosols carrying microspheres, Bacillus globigii spores and M. bovis BCG, concentrating these microparticles onto a collector piece designed for subsequent detection analysis. The unit was also successfully operated in the complex setting of a prison hotspot, enabling detection of a molecular signature for M. tuberculosis in the cough of inmates. Future deployment of this device may lead to a measurable impact on TB case-finding by screening individuals through the aerosols they generate.

Diet change affects intestinal microbiota restoration and improves vertical sleeve gastrectomy outcome in diet-induced obese rats.

PURPOSE: Obesity, a worldwide health problem, is linked to an abnormal gut microbiota and is currently most effectively treated by bariatric surgery. Our aim was to characterize the microbiota of high-fat fed Sprague-Dawley rats when subjected to bariatric surgery (i.e., vertical sleeve gastrectomy) and posterior refeeding with either a high-fat or control diet. We hypothesized that bariatric surgery followed by the control diet was more effective in reverting the microbiota modifications caused by the high-fat diet when compared to either of the two factors alone. METHODS: Using next-generation sequencing of ribosomal RNA amplicons, we analyzed and compared the composition of the cecal microbiota after vertical sleeve gastrectomy with control groups representing non-operated rats, control fed, high-fat fed, and post-operative diet-switched animals. Rats were fed either a high-fat or control low-fat diet and were separated into three comparison groups after eight weeks comprising no surgery, sham surgery, and vertical sleeve gastrectomy. Half of the rats were then moved from the HFD to the control diet. Using next-generation sequencing of ribosomal RNA amplicons, we analyzed the composition of the cecal microbiota of rats allocated to the vertical sleeve gastrectomy group and compared it to that of the non-surgical, control fed, high-fat fed, and post-operative diet-switched groups. Additionally, we correlated different biological parameters with the genera exhibiting the highest variation in abundance between the groups. RESULTS: The high-fat diet was the strongest driver of altered taxonomic composition, relative microbial abundance, and diversity in the cecum. These effects were partially reversed in the diet-switched cohort, especially when combined with sleeve gastrectomy, resulting in increased diversity and shifting relative abundances. Several highly-affected genera were correlated with obesity-related parameters. CONCLUSIONS: The dysbiotic state caused by high-fat diet was improved by the change to the lower fat, higher fiber control diet. Bariatric surgery contributed significantly and additively to the diet in restoring microbiome diversity and complexity. These results highlight the importance of dietary intervention following bariatric surgery for improved restoration of cecal diversity, as neither surgery nor change of diet alone had the same effects as when combined.

The subway microbiome: seasonal dynamics and direct comparison of air and surface bacterial communities.

BACKGROUND: Mass transit environments, such as subways, are uniquely important for transmission of microbes among humans and built environments, and for their ability to spread pathogens and impact large numbers of people. In order to gain a deeper understanding of microbiome dynamics in subways, we must identify variables that affect microbial composition and those microorganisms that are unique to specific habitats. METHODS: We performed high-throughput 16S rRNA gene sequencing of air and surface samples from 16 subway stations in Oslo, Norway, across all four seasons. Distinguishing features across seasons and between air and surface were identified using random forest classification analyses, followed by in-depth diversity analyses. RESULTS: There were significant differences between the air and surface bacterial communities, and across seasons. Highly abundant groups were generally ubiquitous; however, a large number of taxa with low prevalence and abundance were exclusively present in only one sample matrix or one season. Among the highly abundant families and genera, we found that some were uniquely so in air samples. In surface samples, all highly abundant groups were also well represented in air samples. This is congruent with a pattern observed for the entire dataset, namely that air samples had significantly higher within-sample diversity. We also observed a seasonal pattern: diversity was higher during spring and summer. Temperature had a strong effect on diversity in air but not on surface diversity. Among-sample diversity was also significantly associated with air/surface, season, and temperature. CONCLUSIONS: The results presented here provide the first direct comparison of air and surface bacterial microbiomes, and the first assessment of seasonal variation in subways using culture-independent methods. While there were strong similarities between air and surface and across seasons, we found both diversity and the abundances of certain taxa to differ. This constitutes a significant step towards understanding the composition and dynamics of bacterial communities in subways, a highly important environment in our increasingly urbanized and interconnect world. Video abstract.

Pharmacodynamic considerations of collateral sensitivity in design of antibiotic treatment regimen.

INTRODUCTION: Antibiotics have greatly reduced the morbidity and mortality due to infectious diseases. Although antibiotic resistance is not a new problem, its breadth now constitutes a significant threat to human health. One strategy to help combat resistance is to find novel ways to use existing drugs, even those that display high rates of resistance. For the pathogens Escherichia coli and Pseudomonas aeruginosa, pairs of antibiotics have been identified for which evolution of resistance to drug A increases sensitivity to drug B and vice versa. These research groups have proposed cycling such pairs to treat infections, and similar treatment strategies are being investigated for various cancer forms as well. While an exciting treatment prospect, no cycling experiments have yet been performed with consideration of pharmacokinetics and pharmacodynamics. To test the plausibility of such schemes and optimize them, we create a mathematical model with explicit pharmacokinetic/pharmacodynamic considerations. MATERIALS AND METHODS: We evaluate antibiotic cycling protocols using pairs of such antibiotics and investigate the speed of ascent of multiply resistant mutants. RESULTS: Our analyses show that when using low concentrations of antibiotics, treatment failure will always occur due to the rapid ascent and fixation of resistant mutants. However, moderate to high concentrations of some combinations of bacteriostatic and bactericidal antibiotics with multiday cycling prevent resistance from developing and increase the likelihood of treatment success. CONCLUSION: Our results call for guarded optimism in application and development of such treatment protocols.

Evidence for selective bacterial community structuring on microplastics.

In aquatic ecosystems, microplastics are a relatively new anthropogenic substrate that can readily be colonized by biofilm-forming organisms. To examine the effects of substrate type on microbial community assembly, we exposed ambient Baltic bacterioplankton to plastic substrates commonly found in marine environments (polyethylene, polypropylene and polystyrene) as well as native (cellulose) and inert (glass beads) particles for 2 weeks under controlled conditions. The source microbial communities and those of the biofilms were analyzed by Illumina sequencing of the 16S rRNA gene libraries. All biofilm communities displayed lower diversity and evenness compared with the source community, suggesting substrate-driven selection. Moreover, the plastics-associated communities were distinctly different from those on the non-plastic substrates. Whereas plastics hosted greater than twofold higher abundance of Burkholderiales, the non-plastic substrates had a significantly higher proportion of Actinobacteria and Cytophagia. Variation in the community structure, but not the cell abundance, across the treatments was strongly linked to the substrate hydrophobicity. Thus, microplastics host distinct bacterial communities, at least during early successional stages.

Early-Life Human Microbiota Associated With Childhood Allergy Promotes the T Helper 17 Axis in Mice.

The intestinal microbiota influences immune maturation during childhood, and is implicated in early-life allergy development. However, to directly study intestinal microbes and gut immune responses in infants is difficult. To investigate how different types of early-life gut microbiota affect immune development, we collected fecal samples from children with different allergic heredity (AH) and inoculated germ-free mice. Immune responses and microbiota composition were evaluated in the offspring of these mice. Microbial composition in the small intestine, the cecum and the colon were determined by 16S rRNA sequencing. The intestinal microbiota differed markedly between the groups of mice, but only exposure to microbiota associated with AH and known future allergy in children resulted in a T helper 17 (Th17)-signature, both systemically and in the gut mucosa in the mouse offspring. These Th17 responses could be signs of a particular microbiota and a shift in immune development, ultimately resulting in an increased risk of allergy.

Safety and efficacy of composite collagen-silver nanoparticle hydrogels as tissue engineering scaffolds.

The increasing number of multidrug resistant bacteria has revitalized interest in seeking alternative sources for controlling bacterial infection. Silver nanoparticles (AgNPs), are amongst the most promising candidates due to their wide microbial spectrum of action. In this work, we report on the safety and efficacy of the incorporation of collagen coated AgNPs into collagen hydrogels for tissue engineering. The resulting hybrid materials at [AgNPs] < 0.4 µM retained the mechanical properties and biocompatibility for primary human skin fibroblasts and keratinocytes of collagen hydrogels; they also displayed remarkable anti-infective properties against S. aureus, S. epidermidis, E. coli and P. aeruginosa at considerably lower concentrations than silver nitrate. Further, subcutaneous implants of materials containing 0.2 µM AgNPs in mice showed a reduction in the levels of IL-6 and other inflammation markers (CCL24, sTNFR-2, and TIMP1). Finally, an analysis of silver contents in implanted mice showed that silver accumulation primarily occurred within the tissue surrounding the implant.

Persistence: a copacetic and parsimonious hypothesis for the existence of non-inherited resistance to antibiotics.

We postulate that phenotypic resistance to antibiotics, persistence, is not an evolved (selected-for) character but rather like mutation, an inadvertent product of different kinds of errors and glitches. The rate of generation of these errors is augmented by exposure to these drugs. The genes that have been identified as contributing to the production of persisters are analogous to the so-called mutator genes; they modulate the rate at which these errors occur and/or are corrected. In theory, these phenotypically resistant bacteria can retard the rate of microbiological cure by antibiotic treatment.

Anti-peroxyl radical quality and antibacterial properties of rooibos infusions and their pure glycosylated polyphenolic constituents.

The anti-peroxyl radical quality of two aqueous rooibos infusions and solutions of their most abundant glycosylated polyphenols was evaluated using pyrogallol red and fluorescein-based oxygen radical absorbance ratios. It was observed that the artificial infusions, prepared using only the most abundant polyphenols present in rooibos and at concentrations similar to those found in the natural infusions, showed greater antioxidant quality than the latter infusions, reaching values close to those reported for tea infusions. Additionally, the antimicrobial activity of the natural and artificial infusions was assessed against three species of bacteria: Gram (+) Staphylococus epidermidis and Staphylococcus aureus and Gram (-) Escherichia coli. When compared to the natural infusions the artificial beverages did not demonstrate any bacterostatic/cidal activity, suggesting that the antibacterial activity of rooibos is related to compounds other than the glycosylated polyphenols employed in our study.

Nurture your scientific curiosity early in your research career.

Uncertainty makes scientific research challenging and at the same time exciting. Whereas curiosity and passion for uncovering the unknown drive future generations of researchers, the landscape of science has changed. We investigated whether the requirements for having a successful research career are changing, and whether junior researchers are aware of these requirements. Structured discussion with peers and more experienced researchers can point the way forward to an excellent career.

Staphylococcus aureus in continuous culture: a tool for the rational design of antibiotic treatment protocols.

In vitro measures of the pharmacodynamics of antibiotics that account for the factors anticipated for bacteria in infected patients are central to the rational design of antibiotic treatment protocols. We consider whether or not continuous culture devices are a way to obtain these measures. Staphylococcus aureus PS80 in high-density continuous cultures were exposed to oxacillin, ciprofloxacin, vancomycin, gentamicin, daptomycin and linezolid. Contrary to results from low density retentostats as well as to predictions of traditional PK/MIC ratios, daily dosing with up to 100× MIC did not clear these cultures. The densities of S. aureus in these cultures oscillated with constant amplitude and never fell below 10(5) CFU per ml. Save for daptomycin "treated" populations, the densities of bacteria in these cultures remained significantly below that of similar antibiotic-free cultures. Although these antibiotics varied in their pharmacodynamic properties there were only modest differences in their mean densities. Mathematical models and experiments suggest that the dominant factor preventing clearance was wall-adhering subpopulations reseeding the planktonic population which can be estimated and corrected for. Continuous cultures provide a way to evaluate the potential efficacy of antibiotic treatment regimes in vitro under conditions that are more clinically realistic and comprehensive than traditional in vitro PK/PD indices.

Intravital models of infection lay the foundation for tissue microbiology.

In complex environments, such as those found in the human host, pathogenic bacteria constantly battle the unfavorable conditions imposed by the host response to their presence. During Escherichia coli-induced pyelonephritis, a cascade of events are shown in an intravital animal model to occur in a timely and sequential manner, representing the dynamic interplay between host and pathogen. Today, intravital techniques allow for observing infection in the living host. At resolutions almost on the single-cell level, improved detection methods offer a movie-like description of infection dynamics. Tissue microbiology involves monitoring host-pathogen interaction within the dynamic microecology of infectious sites in the live host. This new field holds great promise for insightful research into microbial disease intervention strategies.