Fast identification of <i>Escherichia coli</i> in urinary tract infections using a virulence gene based PCR approach in a novel thermal cycler

Uropathogenic Escherichia coli (UPEC) is the most common causal agent of urinary tract infections (UTIs) in humans. Currently, clinical detection methods take hours (dipsticks) to days (culturing methods), limiting rapid intervention. As an alternative, the use of molecular methods could improve speed and accuracy, but their applicability is complicated by high genomic variability within UPEC strains. Here, we describe a novel PCR-based method for the identification of E. coli in urine. Based on in silico screening of UPEC genomes, we selected three UPEC-specific genes predicted to be involved in pathogenesis (c3509, c3686 (yrbH) and chuA), and one E. coli-specific marker gene (uidA). We validated the method on 128 clinical (UTI) strains. Despite differential occurrences of these genes in uropathogenic E. coli, the method, when using multi-gene combinations, specifically detected the target organism across all samples. The lower detection limit, assessed with model UPEC strains, was approximately 104 CFU/ml. Additionally, the use of this method in a novel ultrafast PCR thermal cycler (Nextgen PCR) allowed a detection time from urine sampling to identification of only 52 min. This is the first study that uses such defined sets of marker genes for the detection of E. coli in UTIs. In addition, we are the first to demonstrate the potential of the Nextgen thermal cycler. Our E. coli identification method has the potential to be a rapid, reliable and inexpensive alternative for traditional methods

Gut-microbiome composition in response to phenylketonuria depends on dietary phenylalanine in BTBR Pah^enu2 mice

Phenylketonuria (PKU) is a metabolic disorder caused by a hepatic enzyme deficiency causing high blood and brain levels of the amino acid Phenylalanine (Phe), leading to severe cognitive and psychological deficits that can be prevented, but not completely, by dietary treatment. The behavioral outcome of PKU could be affected by the gut-microbiome-brain axis, as diet is one of the major drivers of the gut microbiome composition. Gut-microbiome alterations have been reported in treated patients with PKU, although the question remains whether this is due to PKU, the dietary treatment, or their interaction. We, therefore, examined the effects of dietary Phe restriction on gut-microbiome composition and relationships with behavioral outcome in mice. Male and female BTBR Pah(enu2) mice received either a control diet (normal protein, “high” Phe), liberalized Phe-restricted (33% natural protein restriction), or severe Phe-restricted (75% natural protein restriction) diet with protein substitutes for 10 weeks (n = 14 per group). Their behavioral performance was examined in an open field test, novel and spatial object location tests, and a balance beam. Fecal samples were collected and sequenced for the bacterial 16S ribosomal RNA (rRNA) region. Results indicated that PKU on a high Phe diet reduced Shannon diversity significantly and altered the microbiome composition compared with wild-type animals. Phe-restriction prevented this loss in Shannon diversity but changed community composition even more than the high-Phe diet, depending on the severity of the restriction. Moreover, on a taxonomic level, we observed the highest number of differentially abundant genera in animals that received 75% Phe-restriction. Based on correlation analyses with differentially abundant taxa, the families Entereococacceae, Erysipelotrichaceae, Porphyromonadaceae, and the genus Alloprevotella showed interesting relationships with either plasma Phe levels and/or object memory. According to our results, these bacterial taxa could be good candidates to start examining the microbial metabolic potential and probiotic properties in the context of PKU. We conclude that PKU leads to an altered gut microbiome composition in mice, which is least severe on a liberalized Phe-restricted diet. This may suggest that the current Phe-restricted diet for PKU patients could be optimized by taking dietary effects on the microbiome into account

Taxonomic shifts in arbuscular mycorrhizal fungal communities with shade and soil nitrogen across conventionally managed and organic coffee agroecosystems

The composition of arbuscular mycorrhizal fungal (AMF) communities should reflect not only responses to host and soil environments, but also differences in functional roles and costs vs. benefits among arbuscular mycorrhizal fungi. The coffee agroecosystem allows exploration of the effects of both light and soil fertility on AMF communities, because of the variation in shade and soil nutrients farmers generate through field management. We used high-throughput ITS2 sequencing to characterize the AMF communities of coffee roots in 25 fields in Costa Rica that ranged from organic management with high shade and no chemical fertilizers to conventionally managed fields with minimal shade and high N fertilization, and examined relationships between AMF communities and soil and shade parameters with partial correlations, NMDS, PERMANOVA, and partial least squares analysis. Gigasporaceae and Acaulosporaceae dominated coffee AMF communities in terms of relative abundance and richness, respectively. Gigasporaceae richness was greatest in conventionally managed fields, while Glomeraceae richness was greatest in organic fields. While total AMF richness and root colonization did not differ between organic and conventionally managed fields, AMF community composition did; these differences were correlated with soil nitrate and shade. OTUs differing in relative abundance between conventionally managed and organic fields segregated into four groups: Gigasporaceae associated with high light and nitrate availability, Acaulosporaceae with high light and low nitrate availability, Acaulosporaceae and a single relative of Rhizophagus fasciculatus with shade and low nitrate availability, and Claroideoglomus/Glomus with conventionally managed fields but uncorrelated with shade and soil variables. The association of closely related taxa with similar shade and light availabilities is consistent with phylogenetic trait conservatism in AM fungi

Contrasting patterns of functional diversity in coffee root fungal communities associated with organic and conventionally managed fields

The structure and function of fungal communities in the coffee rhizosphere are influenced by crop environment. Because coffee can be grown along a management continuum from conventional application of pesticides and fertilizers in full sun to organic management in a shaded understory, we used coffee fields to hold host constant while comparing rhizosphere fungal communities under markedly different environmental conditions with regard to shade and inputs. We characterized the shade and soil environment in 25 fields under conventional, organic, or transitional management in two regions of Costa Rica. We amplified the internal transcribed spacer 2 (ITS2) region of fungal DNA from coffee roots in these fields and characterized the rhizosphere fungal community via high-throughput sequencing. Sequences were assigned to guilds to determine differences in functional diversity and trophic structure among coffee field environments. Organic fields had more shade, a greater richness of shade tree species, and more leaf litter and were less acidic, with lower soil nitrate availability and higher soil copper, calcium, and magnesium availability than conventionally managed fields, although differences between organic and conventionally managed fields in shade and calcium and magnesium availability depended on region. Differences in richness and community composition of rhizosphere fungi between organic and conventionally managed fields were also correlated with shade, soil acidity, and nitrate and copper availability. Trophic structure differed with coffee field management. Saprotrophs, plant pathogens, and mycoparasites were more diverse, and plant pathogens were more abundant, in organic than in conventionally managed fields, while saprotroph-plant pathogens were more abundant in conventionally managed fields. These differences reflected environmental differences and depended on region

SARS-CoV-2 Omicron is an immune escape variant with an altered cell entry pathway

Vaccines based on the spike protein of SARS-CoV-2 are a cornerstone of the public health response to COVID-19. The emergence of hypermutated, increasingly transmissible variants of concern (VOCs) threaten this strategy. Omicron (B.1.1.529), the fifth VOC to be described, harbours multiple amino acid mutations in spike, half of which lie within the receptor-binding domain. Here we demonstrate substantial evasion of neutralization by Omicron BA.1 and BA.2 variants in vitro using sera from individuals vaccinated with ChAdOx1, BNT162b2 and mRNA-1273. These data were mirrored by a substantial reduction in real-world vaccine effectiveness that was partially restored by booster vaccination. The Omicron variants BA.1 and BA.2 did not induce cell syncytia in vitro and favoured a TMPRSS2-independent endosomal entry pathway, these phenotypes mapping to distinct regions of the spike protein. Impaired cell fusion was determined by the receptor-binding domain, while endosomal entry mapped to the S2 domain. Such marked changes in antigenicity and replicative biology may underlie the rapid global spread and altered pathogenicity of the Omicron variant