The bacterial density of clinical rectal swabs is highly variable, correlates with sequencing contamination, and predicts patient risk of extraintestinal infection

Background In ecology, population density is a key feature of community analysis. Yet in studies of the gut microbiome, bacterial density is rarely reported. Studies of hospitalized patients commonly use rectal swabs for microbiome analysis, yet variation in their bacterial density—and the clinical and methodologic significance of this variation—remains undetermined. We used an ultra-sensitive quantification approach—droplet digital PCR (ddPCR)—to quantify bacterial density in rectal swabs from 118 hospitalized patients. We compared bacterial density with bacterial community composition (via 16S rRNA amplicon sequencing) and clinical data to determine if variation in bacterial density has methodological, clinical, and prognostic significance. Results Bacterial density in rectal swab specimens was highly variable, spanning five orders of magnitude (1.2 × 104–3.2 × 109 16S rRNA gene copies/sample). Low bacterial density was strongly correlated with the detection of sequencing contamination (Spearman ρ = − 0.95, p < 10−16). Low-density rectal swab communities were dominated by peri-rectal skin bacteria and sequencing contaminants (p < 0.01), suggesting that some variation in bacterial density is explained by sampling variation. Yet bacterial density was also associated with important clinical exposures, conditions, and outcomes. Bacterial density was lower among patients who had received piperacillin-tazobactam (p = 0.017) and increased among patients with multiple medical comorbidities (Charlson score, p = 0.0040) and advanced age (p = 0.043). Bacterial density at the time of hospital admission was independently associated with subsequent extraintestinal infection (p = 0.0028), even when controlled for severity of illness and comorbidities. Conclusions The bacterial density of rectal swabs is highly variable, and this variability is of methodological, clinical, and prognostic significance. Microbiome studies using rectal swabs are vulnerable to sequencing contamination and should include appropriate negative sequencing controls. Among hospitalized patients, gut bacterial density is associated with clinical exposures (antibiotics, comorbidities) and independently predicts infection risk. Bacterial density is an important and under-studied feature of gut microbiome community analysis.

Virulence potential of a multidrug-resistant Escherichia coli strain belonging to the emerging clonal group ST101-B1 isolated from bloodstream infection

Escherichia coli EC121 is a multidrug-resistant (MDR) strain isolated from bloodstream infection of an inpatient with persistent gastroenteritis and Zone T lymphoma, that died due to septic shock. Despite causing an extraintestinal infection, it harbors few known virulence factors and was assigned into phylogenetic group B1. To evaluate if the EC121 was pathogenic or opportunistic, its genome was sequenced, and an in vitro characterization of some pathogenicity-associated properties was performed. The data retrieved from genome analysis showed that E. coli strain EC121 belongs to the O154:H25 serotype, and to ST101-B1, which was epidemiologically linked to extraintestinal infections and antimicrobial resistance spread as well. Moreover, it was closely related to Shiga-toxin producing E. coli (STEC). Besides, strain EC121 is an MDR strain harboring 14 antimicrobial resistance genes, including blaCTX-M-2, and more than 50 complete virulence genetic clusters, which are reported to be associated either with DEC or ExPEC. The strain also displays the capacity to adhere to a variety of cell lineages, and invade T24 bladder cells, as well as the ability to form biofilms on abiotic surfaces, and survive the bactericidal serum complement activity. Additionally, it is virulent in the Galleria mellonella model. Altogether, E. coli EC121 unveiled to be a pathogen powered by its multi-drug resistance characteristic. Carry out studies providing accurate information about the virulence potential of all kinds of MDR strains are essential because these studies will help in the development of alternative therapies of infection management and spread control of MDR strains.Authors summaryThe phylogenetic origin of extraintestinal pathogenic Escherichia coli is mostly associated with phylogroup B2, and the majority of the studies regarding extraintestinal infection focus on the most virulent strains, which might also present multidrug-resistant (MDR) phenotype. Strains belonging to phylogroup B1 and isolated from extraintestinal infections are considered as opportunist pathogens and have their virulence neglected. We focus our study in one MDR strain isolated from bloodstream infection that belongs to phylogenetic group B1 to enlarge the knowledge about the virulence of this kind of strain. We demonstrated that the EC121 is capable of adheres to intestinal and bladder human cells, and invades the latter one; it survives to human serum bactericidal effects and produces biofilm. Additionally, the in vivo assay confirmed the EC121 virulence, showing that it should be considered a pathogenic strain. The genetic analyzes highlighted important aspects of EC121 which are typical from strains of sequence type 101, like its involvement in the spread of antimicrobial resistance genes and its relationship with extraintestinal infection from diverse sources. Information concerning the virulence of MDR strains is important for the development of global actions treating the spread of antimicrobial resistance, as well as to elucidate the pathogenesis of strains that were considered as an opportunist.

Fine-Scale Structure Analysis Shows Epidemic Patterns of Clonal Complex 95, a Cosmopolitan Escherichia coli Lineage Responsible for Extraintestinal Infection

ABSTRACT Escherichia coli clonal complex 95 represents a cosmopolitan, genetically diverse lineage, and the extensive substructure observed in this lineage is epidemiologically and clinically relevant. The frequency with which CC95 strains are responsible for extraintestinal infection appears to have been stable over the past 15 years. However, the different subgroups identified within this lineage have an epidemic structure depending on the host, sample, continent, and time. Thus, the evolution and spread of strains belonging to CC95 are very different from those of another cosmopolitan human-associated clonal complex, CC131, which has increased significantly in frequency as a cause of extraintestinal infection over the past 15 years due to the evolution and spread of two very closely related, nearly monomorphic lineages. The Escherichia coli lineage known as clonal complex 95 (CC95) is a cosmopolitan human-associated lineage responsible for a significant fraction of extraintestinal infections of humans. Whole-genome sequence data of 200 CC95 strains from various origins enabled determination of the CC95 pangenome. The pangenome analysis revealed that strains of the complex could be assigned to one of five subgroups that vary in their serotype, extraintestinal virulence, virulence gene content, and antibiotic resistance gene profile. A total of 511 CC95 strains isolated from humans living in France, Australia, and the United States were screened for their subgroup membership using a PCR-based method. The CC95 subgroups are nonrandomly distributed with respect to their geographic origin. The relative frequency of the subgroups was shown to change through time, although the nature of the changes varies with continent. Strains of the subgroups are also nonrandomly distributed with respect to source of isolation (blood, urine, or feces) and host sex. Collectively, the evidence indicates that although strains belonging to CC95 may be cosmopolitan, human movement patterns have been insufficient to homogenize the distribution of the CC95 subgroups. Rather, the manner in which CC95 strains evolve appears to vary both spatially and temporally. Although CC95 strains appeared globally as pandemic, fine-scale structure analysis shows epidemic patterns of the CC95 subgroups. Furthermore, the observation that the relative frequency of CC95 subgroups at a single locality has changed over time indicates that the relative fitness of the subgroups has changed. IMPORTANCE Escherichia coli clonal complex 95 represents a cosmopolitan, genetically diverse lineage, and the extensive substructure observed in this lineage is epidemiologically and clinically relevant. The frequency with which CC95 strains are responsible for extraintestinal infection appears to have been stable over the past 15 years. However, the different subgroups identified within this lineage have an epidemic structure depending on the host, sample, continent, and time. Thus, the evolution and spread of strains belonging to CC95 are very different from those of another cosmopolitan human-associated clonal complex, CC131, which has increased significantly in frequency as a cause of extraintestinal infection over the past 15 years due to the evolution and spread of two very closely related, nearly monomorphic lineages.