Genome wide association study of human bacteremia Escherichia coli isolates identifies genetic determinants for the portal of entry but not fatal outcome

Escherichia coli is an important cause of bloodstream infections (BSI), which is of concern given its high mortality and increasing worldwide prevalence. Finding bacterial genetic variants that might contribute to patient death is of interest to better understand its mechanism and implement diagnostic methods that specifically look for those factors. E. coli samples isolated from patients with BSI are an ideal dataset to systematically search for those variants, as long as the influence of host factors such as comorbidities are taken into account. Here we performed a genome-wide association study (GWAS) using data from 910 patients with E. coli BSI from hospitals in Paris, France; we looked for associations between bacterial genetic variants and three patient outcomes (death at 28 days, septic shock and admission to intensive care unit), as well as two portals of entry (urinary and digestive tract), using various clinical variables from each patient to account for host factors. We did not find any associations between genetic variants and patient outcomes, potentially confirming the strong influence of host factors in influencing the course of BSI; we however found a strong association between the papGII/papGIII operon and entrance of E. coli through the urinary tract, which demonstrates the power of bacterial GWAS even when applied to actual clinical data. Despite the lack of associations between E. coli genetic variants and patient outcomes, we estimate that increasing the sample size by one order of magnitude could lead to the discovery of some putative causal variants. The adoption of bacterial genome sequencing of clinical isolates might eventually lead to the elucidation of the mechanisms behind BSI progression and the development of sequence-based diagnostics.

Perna canaliculus as an Ecological Material in the Removal of o-Cresol Pollutants from Soil

Soil contamination with cresol is a problem of the 21st century and poses a threat to soil microorganisms, humans, animals, and plants. The lack of precise data on the potential toxicity of o-cresol in soil microbiome and biochemical activity, as well as the search for effective remediation methods, inspired the aim of this study. Soil is subjected to four levels of contamination with o-cresol: 0, 0.1, 1, 10, and 50 mg o-cresol kg−1 dry matter (DM) of soil and the following are determined: the count of eight groups of microorganisms, colony development index (CD) and ecophysiological diversity index (EP) for organotrophic bacteria, actinobacteria and fungi, and the bacterial genetic diversity. Moreover, the responses of seven soil enzymes are investigated. Perna canaliculus is a recognized biosorbent of organic pollutants. Therefore, microbial biostimulation with Perna canaliculus shells is used to eliminate the negative effect of the phenolic compound on the soil microbiome. Fungi appears to be the microorganisms most sensitive to o-cresol, while Pseudomonas sp. is the least sensitive. In o-cresol-contaminated soils, the microbiome is represented mainly by the bacteria of the Proteobacteria and Firmicutes phyla. Acid phosphatase, alkaline phosphatase and urease can be regarded as sensitive indicators of soil disturbance. Perna canaliculus shells prove to be an effective biostimulator of soil under pressure with o-cresol.

Mediterranean Spontaneously Fermented Sausages: Spotlight on Microbiological and Quality Features to Exploit Their Bacterial Biodiversity

The wide array of spontaneously fermented sausages of the Mediterranean area can represent a reservoir of microbial biodiversity and can be an important source of new technological and functional strains able to preserve product properties, counteracting the impoverishment of their organoleptic typical features due to the introduction of commercial starter cultures. We analysed 15 artisanal salamis from Italy, Spain, Croatia and Slovenia to evaluate the microbiota composition, through culture-dependent and culture-independent techniques (i.e., metagenomic analysis), chemical–physical features, biogenic amines and aroma profile. The final pH varied according to origin and procedures (e.g., higher pH in Italian samples due to long ripening and mold growth). Lactic acid bacteria (LAB) and coagulase-negative cocci (CNC) were the dominant population, with highest LAB counts in Croatian and Italian samples. Metagenomic analysis showed high variability in qualitative and quantitative microbial composition: among LAB, Latilactobacillus sakei was the dominant species, but Companilactobacillus spp. was present in high amounts (45–55% of the total ASVs) in some Spanish sausages. Among staphylococci, S. epidermidis, S. equorum, S. saprophyticus, S. succinus and S. xylosus were detected. As far as biogenic amines, tyramine was always present, while histamine was found only in two Spanish samples. These results can valorize the bacterial genetic heritage present in Mediterranean products, to find new candidates of autochthonous starter cultures or bioprotective agents.

Genome-wide association studies reveal the role of polymorphisms affecting factor H binding protein expression in host invasion by Neisseria meningitidis

Many invasive bacterial diseases are caused by organisms that are ordinarily harmless components of the human microbiome. Effective interventions against these microbes require an understanding of the processes whereby symbiotic or commensal relationships transition into pathology. Here, we describe bacterial genome-wide association studies (GWAS) of Neisseria meningitidis, a common commensal of the human respiratory tract that is nevertheless a leading cause of meningitis and sepsis. An initial GWAS discovered bacterial genetic variants, including single nucleotide polymorphisms (SNPs), associated with invasive meningococcal disease (IMD) versus carriage in several loci across the meningococcal genome, encoding antigens and other extracellular components, confirming the polygenic nature of the invasive phenotype. In particular, there was a significant peak of association around the fHbp locus, encoding factor H binding protein (fHbp), which promotes bacterial immune evasion of human complement by recruiting complement factor H (CFH) to the meningococcal surface. The association around fHbp with IMD was confirmed by a validation GWAS, and we found that the SNPs identified in the validation affected the 5’ region of fHbp mRNA, altering secondary RNA structures, thereby increasing fHbp expression and enhancing bacterial escape from complement-mediated killing. This finding is consistent with the known link between complement deficiencies and CFH variation with human susceptibility to IMD. These observations demonstrate the importance of human and bacterial genetic variation across the fHbp:CFH interface in determining IMD susceptibility, the transition from carriage to disease.

Predicting the taxonomic and environmental sources of integron gene cassettes using structural and sequence homology of attC sites

Integrons are bacterial genetic elements that can capture mobile gene cassettes. They are mostly known for their role in the spread of antibiotic resistance cassettes, contributing significantly to the global resistance crisis. These resistance cassettes likely originated from sedentary chromosomal integrons, having subsequently been acquired and disseminated by mobilised integrons. However, their taxonomic and environmental origins are unknown. Here, we use cassette recombination sites (attCs) to predict the origins of those resistance cassettes now spread by mobile integrons. We modelled the structure and sequence homology of 1,978 chromosomal attCs from 11 different taxa. Using these models, we show that at least 27% of resistance cassettes have attCs that are structurally conserved among one of three taxa (Xanthomonadales, Spirochaetes and Vibrionales). Indeed, we found some resistance cassettes still residing in sedentary chromosomal integrons of the predicted taxa. Further, we show that attCs cluster according to host environment rather than host phylogeny, allowing us to assign their likely environmental sources. For example, the majority of β-lactamases and aminoglycoside acetyltransferases, the two most prevalent resistance cassettes, appear to have originated from marine environments. Together, our data represent the first evidence of the taxonomic and environmental origins of resistance cassettes spread by mobile integrons.

Insights into evolution and coexistence of the colibactin- and yersiniabactin secondary metabolite determinants in enterobacterial populations

The bacterial genotoxin colibactin interferes with the eukaryotic cell cycle by causing dsDNA breaks. It has been linked to bacterially induced colorectal cancer in humans. Colibactin is encoded by a 54 kb genomic region in Enterobacteriaceae . The colibactin genes commonly co-occur with the yersiniabactin biosynthetic determinant. Investigating the prevalence and sequence diversity of the colibactin determinant and its linkage to the yersiniabactin operon in prokaryotic genomes, we discovered mainly species-specific lineages of the colibactin determinant and classified three main structural settings of the colibactin–yersiniabactin genomic region in Enterobacteriaceae . The colibactin gene cluster has a similar but not identical evolutionary track to that of the yersiniabactin operon. Both determinants could have been acquired on several occasions and/or exchanged independently between enterobacteria by horizontal gene transfer. Integrative and conjugative elements play(ed) a central role in the evolution and structural diversity of the colibactin–yersiniabactin genomic region. Addition of an activating and regulating module (clbAR) to the biosynthesis and transport module (clbB-S) represents the most recent step in the evolution of the colibactin determinant. In a first attempt to correlate colibactin expression with individual lineages of colibactin determinants and different bacterial genetic backgrounds, we compared colibactin expression of selected enterobacterial isolates in vitro. Colibactin production in the tested Klebsiella species and Citrobacter koseri strains was more homogeneous and generally higher than that in most of the Escherichia coli isolates studied. Our results improve the understanding of the diversity of colibactin determinants and its expression level, and may contribute to risk assessment of colibactin-producing enterobacteria.

Comparative analysis of three studies measuring fluorescence from engineered bacterial genetic constructs

Reproducibility is a key challenge of synthetic biology, but the foundation of reproducibility is only as solid as the reference materials it is built upon. Here we focus on the reproducibility of fluorescence measurements from bacteria transformed with engineered genetic constructs. This comparative analysis comprises three large interlaboratory studies using flow cytometry and plate readers, identical genetic constructs, and compatible unit calibration protocols. Across all three studies, we find similarly high precision in the calibrants used for plate readers. We also find that fluorescence measurements agree closely across the flow cytometry results and two years of plate reader results, with an average standard deviation of 1.52-fold, while the third year of plate reader results are consistently shifted by more than an order of magnitude, with an average shift of 28.9-fold. Analyzing possible sources of error indicates this shift is due to incorrect preparation of the fluorescein calibrant. These findings suggest that measuring fluorescence from engineered constructs is highly reproducible, but also that there is a critical need for access to quality controlled fluorescent calibrants for plate readers.

Pathogen clonal expansion underlies multiorgan dissemination and organ-specific outcomes during systemic infection

The dissemination of pathogens through blood and their establishment within organs lead to severe clinical outcomes. However, the within-host dynamics that underly pathogen spread to and clearance from systemic organs remain largely uncharacterized. Here, we investigate the population dynamics of extraintestinal pathogenic E. coli, a common cause of bacteremia, during systemic infection. We show that while bacteria are largely cleared by most organs, organ-specific clearance failures are pervasive and result from dramatic expansions of clones representing less than 0.0001% of the inoculum. Clonal expansion underlies the variability in bacterial burden between animals, and stochastic dissemination of clones profoundly alters the pathogen population structure within organs. Despite variable pathogen expansion events, host bottlenecks are consistent yet highly sensitive to infection variables, including inoculum size and macrophage depletion. Finally, we identify organ-specific bacterial genetic factors that distinguish between establishment of within-organ pathogen populations and subsequent survival or expansion.

Retron Editing for Precise Genome Editing without Exogenous Donor DNA in Human Cells

CRISPR-Cas9 mediated seamless genome editing can be achieved by incorporating donor DNA into the CRISPR-Cas9 target loci via homology-directed repair (HDR), albeit with relative low efficiency due to the inefficient delivery of exogenous DNA. Retrons are bacterial genetic element composed of a non-coding RNA (ncRNA) and reverse transcriptase (RT). Retrons coupled with CRISPR-Cas9 have been shown to enhance precise genome editing via HDR in yeast through fusing guide RNA (gRNA) to the 3′ end of retron ncRNA, producing multicopy single-stranded DNA (msDNA) covalently tethered to gRNA. Here, we further engineered retrons by fusing Cas9 with E.coli RT from different clades and joining gRNA at the 5′ end of retron ncRNA, and found that retron editing can achieve precise genome editing efficiently in human cells. By co- expression of Cas9-RT fusions and retron-ncRNA gRNA (rgRNA) in HEK293T cells, we demonstrated the rates of retron editing at endogenous genomic loci was up to 10 %. We expect our retron editing system could aid in advancing the ex vivo and in vivo therapeutic applications of retron.

Experimental Evolution in Plant-Microbe Systems: A Tool for Deciphering the Functioning and Evolution of Plant-Associated Microbial Communities

In natural environments, microbial communities must constantly adapt to stressful environmental conditions. The genetic and phenotypic mechanisms underlying the adaptive response of microbial communities to new (and often complex) environments can be tackled with a combination of experimental evolution and next generation sequencing. This combination allows to analyse the real-time evolution of microbial populations in response to imposed environmental factors or during the interaction with a host, by screening for phenotypic and genotypic changes over a multitude of identical experimental cycles. Experimental evolution (EE) coupled with comparative genomics has indeed facilitated the monitoring of bacterial genetic evolution and the understanding of adaptive evolution processes. Basically, EE studies had long been done on single strains, allowing to reveal the dynamics and genetic targets of natural selection and to uncover the correlation between genetic and phenotypic adaptive changes. However, species are always evolving in relation with other species and have to adapt not only to the environment itself but also to the biotic environment dynamically shaped by the other species. Nowadays, there is a growing interest to apply EE on microbial communities evolving under natural environments. In this paper, we provide a non-exhaustive review of microbial EE studies done with systems of increasing complexity (from single species, to synthetic communities and natural communities) and with a particular focus on studies between plants and plant-associated microorganisms. We highlight some of the mechanisms controlling the functioning of microbial species and their adaptive responses to environment changes and emphasize the importance of considering bacterial communities and complex environments in EE studies.