Synthesis of current pediatric urinary microbiome research.

The human urinary bladder hosts a complex microbial community of low biomass referred to as the urobiome. While the composition of the urobiome has been investigated in adults for over a decade now, only a few studies have considered the presence and composition of the urobiome in children. It is critical to explore how the urobiome develops throughout the life span and how it changes in the presence of various health conditions. Therefore, we set to review the available data on pediatric urobiome composition and its development with age and disease. In addition, we focused on identifying and reporting specific gaps in our knowledge of the pediatric urobiome that we hope will be addressed by future studies in this swiftly developing field with fast-improving methods and consensus.

The Highly Durable Antibacterial Gel-like Coatings for Textiles.

Hospital-acquired infections are considered a priority for public health systems since they pose a significant burden for society. High-touch surfaces of healthcare centers, including textiles, provide a suitable environment for pathogenic bacteria to grow, necessitating incorporating effective antibacterial agents into textiles. This paper introduces a highly durable antibacterial gel-like solution, Silver Shell™ finish, which contains chitosan-bound silver chloride microparticles. The study investigates the coating's environmental impact, health risks, and durability during repeated washing. The structure of the Silver Shell™ finish was studied using transmission electron microscopy (TEM) and energy-dispersive X-ray spectroscopy (EDX). The TEM images showed a core-shell structure, with chitosan forming a protective shell around groupings of silver microparticles. The field-emission scanning electron microscopy (FESEM) demonstrated the uniform deposition of Silver Shell™ on the surfaces of the fabrics. AATCC Test Method 100 was employed to quantitatively analyze the antibacterial properties of the fabrics coated with silver microparticles. Two types of bacteria, Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli), were used in this study. The antibacterial results showed that after 75 wash cycles, a 100% reduction for both S. aureus and E. coli in the coated samples using crosslinking agents was observed. The coated samples without a crosslinking agent exhibited 99.88% and 99.81% reductions for S. aureus and E. coli after 50 washing cycles. To compare the antibacterial properties toward non-pathogenic and pathogenic strains of the same species, MG1655 model E. coli strain (ATCC 29213) and a multidrug-resistant clinical isolate were used. The results showed the antibacterial efficiency of the Silver ShellTM solution (up to 99.99% reduction) coated on cotton fabric. AATCC-147 was performed to investigate the coated samples' leaching properties and the crosslinking agent's effects against S. aureus and E. coli. All coated samples demonstrated remarkable antibacterial efficacy, even after 75 wash cycles. The crosslinking agent facilitated durable attachment between the silver microparticles and cotton substrate, minimizing the release of particles from the fabrics. Color measurements were conducted to assess the color differences resulting from the coating process. The results indicated fixation values of 44%, 32%, and 28% following 25, 50, and 75 washing cycles, respectively.

Characterization of pediatric urinary microbiome at species-level resolution indicates variation due to sex, age, and urologic history.

BACKGROUND: Recently, associations between recurrent urinary tract infections (UTI) and the urinary microbiome (urobiome) composition have been identified in adults. However, little is known about the urobiome in children. We aimed to characterize the urobiome of children with species-level resolution and to identify associations based on UTI history. STUDY DESIGN: Fifty-four children (31 females and 21 males) from 3 months to 11 years of age participated in the study. Catheterized urine specimens were obtained from children undergoing a clinically indicated voiding cystourethrogram. To improve the analysis of the pediatric urobiome, we used a novel protocol using filters to collect biomass from the urine coupled with synthetic long-read 16S rRNA gene sequencing to obtain culture-independent species-level resolution data. We tested for differences in microbial composition between sex and history of UTIs using non-parametric tests on individual bacteria and alpha diversity measures. RESULTS: We detected bacteria in 61% of samples from 54 children (mean age 40.7 months, 57% females). Similar to adults, urobiomes were distinct across individuals and varied by sex. The urobiome of females showed higher diversity as measured by the inverse Simpson and Shannon indices but not the Pielou evenness index or number of observed species (p = 0.05, p = 0.04, p = 0.35, and p = 0.11, respectively). Additionally, several species were significantly overrepresented in females compared to males, including those from the genera Anaerococcus, Prevotella, and Schaalia (p = 0.03, 0.04, and 0.02, respectively). Urobiome diversity increased with age, driven mainly by males. Comparison of children with a history of 1, 2, or 3+ UTIs revealed that urobiome diversity significantly decreases in the group that experienced 3+ UTIs as measured by the Simpson, Shannon, and Pielou indices (p = 0.03, p = 0.05, p = 0.01). Several bacteria were also found to be reduced in abundance. DISCUSSION: In this study, we confirm that urobiome can be identified from catheter-collected urine specimens in infants as young as 3 months, providing further evidence that the pediatric bladder is not sterile. In addition to confirming variations in the urobiome related to sex, we identify age-related changes in children under 5 years of age, which conflicts with some prior research. We additionally identify associations with a history of UTIs. CONCLUSIONS: Our study provides additional evidence that the pediatric urobiome exists. The bacteria in the bladder of children appear to be affected by early urologic events and warrants future research.

The Highly Durable Antibacterial Gel-like Coatings for Textiles.

Hospital-acquired infections are considered a priority for public health systems, which poses a significant burden for society. High-touch surfaces of healthcare centers, including textiles, provide a suitable environment for pathogenic bacteria to grow, necessitating incorporating effective antibacterial agents into textiles. This paper introduces a highly durable antibacterial gel-like solution, Silver Shell finish, which contains chitosan-bound silver chloride microparticles. The study investigates the coating's environmental impact, health risks, and durability during repeated washing. The structure of the Silver Shell finish was studied using Transmission Electron Microscopy (TEM) and Energy-Dispersive X-ray Spectroscopy (EDX). TEM images showed a core-shell structure, with chitosan forming a protective shell around groupings of silver micro-particles. Field Emission Scanning Electron Microscopy (FESEM) demonstrated the uniform deposition of Silver Shell on the surface of fabrics. AATCC Test Method 100 was employed to quantitatively analyze the antibacterial properties of fabrics coated with silver microparticles. Two types of bacteria, Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) were used in this study. The antibacterial results showed that after 75 wash cycles, a 100% reduction for both S. aureus and E. coli in the coated samples using crosslinking agents was observed. The coated samples without a crosslinking agent exhibited a 99.88% and 99.81% reduction for S. aureus and E. coli after 50 washing cycles. AATCC-147 was performed to investigate the coated samples' leaching properties and the crosslinking agent's effect against S. aureus and E. coli. All coated samples demonstrated remarkable antibacterial efficacy even after 75 wash cycles.

Characterization of pediatric urinary microbiome at species-level resolution indicates variation due to sex, age, and urologic history.

Background: Recently, associations between recurrent urinary tract infections (UTI) and the urinary microbiome (urobiome) composition have been identified in adults. However, little is known about the urobiome in children. We aimed to characterize the urobiome of children with species-level resolution and to identify associations based on UTI history. Study design: Fifty-four children (31 females and 21 males) from 3 months to 5 years of age participated in the study. Catheterized urine specimens were obtained from children undergoing a clinically indicated voiding cystourethrogram. To improve the analysis of the pediatric urobiome, we used a novel protocol using filters to collect biomass from the urine coupled with synthetic long-read 16S rRNA gene sequencing to obtain culture-independent species-level resolution data. We tested for differences in microbial composition between sex and history of UTIs using non-parametric tests on individual bacteria and alpha diversity measures. Results: We detected bacteria in 61% of samples from 54 children (mean age 40.7 months, 57% females). Similar to adults, urobiomes were distinct across individuals and varied by sex. The urobiome of females showed higher diversity as measured by the inverse Simpson and Shannon indices but not the Pielou evenness index or number of observed species (p = 0.05, p=0.04, p = 0.35, and p = 0.11, respectively). Additionally, several species were significantly overrepresented in females compared to males, including those from the genera Anaerococcus, Prevotella, and Schaalia (p = 0.03, 0.04, and 0.02, respectively). Urobiome diversity increased with age, driven mainly by males. Comparison of children with a history of 1, 2, or 3+ UTIs revealed that urobiome diversity significantly decreases in the group that experienced 3+ UTIs as measured by the Simpson, Shannon, and Pielou indices (p = 0.03, p = 0.05, p = 0.01). Several bacteria were also found to be reduced in abundance. Discussion: In this study, we confirm that urobiome can be identified from catheter-collected urine specimens in infants as young as 3 months, providing further evidence that the pediatric bladder is not sterile. In addition to confirming variations in the urobiome related to sex, we identify age-related changes in children under 5 years of age, which conflicts with some prior research. We additionally identify associations with a history of UTIs. Conclusions: Our study provides additional evidence that the pediatric urobiome exists. The bacteria in the bladder of children appear to be affected by early urologic events and warrants future research.

Draft genomes of Lactobacillus delbrueckii and Klebsiella pneumoniae coexisting within a female urinary bladder.

Here, we present the draft genome sequences of Lactobacillus delbrueckii and Klebsiella pneumoniae, both isolated from the urinary bladder of an asymptomatic post-menopausal female patient with a diagnosis of recurrent urinary tract infections. These genomes will facilitate analyses of interbacterial interactions in the urinary microbiome.

Genome Sequences of Bacteria Isolated from the International Space Station Water Systems.

We report draft genomes of five bacteria recovered from the U.S. and Russian water systems onboard the International Space Station. The five genera include Ralstonia, Burkholderia, Cupriavidus, Methylobacterium, and Pseudomonas. These sequences will help further the understanding of water reclamation and environmental control and life support systems in space.

Genome Sequences of Three Common Bacterial Isolates from Wastewater from the Water Processor Assembly at the International Space Station.

Bacteria from the genera Burkholderia, Ralstonia, and Methylobacterium were consistently detected in water of the life support systems at the International Space Station. Here, we report complete genomes of recent isolates that are representative of these genera to support future studies in biofilm and wastewater treatment in space habitats.

Mitigation and use of biofilms in space for the benefit of human space exploration.

Biofilms are self-organized communities of microorganisms that are encased in an extracellular polymeric matrix and often found attached to surfaces. Biofilms are widely present on Earth, often found in diverse and sometimes extreme environments. These microbial communities have been described as recalcitrant or protective when facing adversity and environmental exposures. On the International Space Station, biofilms were found in human-inhabited environments on a multitude of hardware surfaces. Moreover, studies have identified phenotypic and genetic changes in the microorganisms under microgravity conditions including changes in microbe surface colonization and pathogenicity traits. Lack of consistent research in microgravity-grown biofilms can lead to deficient understanding of altered microbial behavior in space. This could subsequently create problems in engineered systems or negatively impact human health on crewed spaceflights. It is especially relevant to long-term and remote space missions that will lack resupply and service. Conversely, biofilms are also known to benefit plant growth and are essential for human health (i.e., gut microbiome). Eventually, biofilms may be used to supply metabolic pathways that produce organic and inorganic components useful to sustaining life on celestial bodies beyond Earth. This article will explore what is currently known about biofilms in space and will identify gaps in the aerospace industry's knowledge that should be filled in order to mitigate or to leverage biofilms to the advantage of spaceflight.

Commensal Urinary Lactobacilli Inhibit Major Uropathogens In Vitro With Heterogeneity at Species and Strain Level.

The human urinary microbiome is thought to affect the development and progression of urinary tract infections (UTI), particularly recurrent UTIs in aging populations of women. To understand the possible interactions of urinary pathogens with commensal bacteria inhabiting the aging bladder, we conducted an initial functional assessment of a representative set of urinary lactobacilli that dominate this niche in postmenopausal women. We created a repository of urinary bladder bacteria isolated via Enhanced Quantitative Urinary Culture (EQUC) from healthy postmenopausal women, as well as those with a culture-proven recurrent UTI (rUTI) diagnosis. This repository contains lactobacilli strains from eight different species. As many other lactobacilli are known to inhibit human pathogens, we hypothesized that some urinary lactobacilli will have similar abilities to inhibit the growth of typical uropathogens and thus, provide a link between the urinary microbiome and the predisposition to the rUTI. Therefore, we screened the urinary lactobacilli in our repository for their ability to inhibit model uropathogens in vitro. We observed that many urinary isolates strongly inhibit model strains of gram-negative Escherichia coli and Klebsiella pneumoniae but demonstrate less inhibition of gram-positive Enterococcus faecalis. The observed inhibition affected model strains of uropathogens as well as clinical and multidrug-resistant isolates of those species. Our preliminary analysis of inhibition modes suggests a combination of pH-dependent and cell-dependent inhibition. Overall, inhibition strongly varies among species and strains of urinary lactobacilli. While the strength of the inhibition is not predictive of health outcomes in this limited repository, there is a high level of species and strain diversity that warrants future detailed investigations.

Draft Genome Sequence of a Lactobacillus gasseri Strain Isolated from the Catheterized Urine of a Healthy Postmenopausal Woman.

Urinary microbiome composition has been found to associate with health status and to change with age. Lactobacillus gasseri is one of the most frequently found lactic acid bacteria in the vaginal and urinary tracts of women. Here, we report a draft genome sequence of a urinary L. gasseri strain isolated from a healthy postmenopausal woman.

Complete sequence of classic F-type plasmid pRK100 shows unique conservation over time and geographic location.

Plasmids exhibit great diversity of gene content and host ranges and are famous for quick adaptation to the genetic background of the bacterial host cell. In addition to observing ever evolving plasmids, some plasmids have conserved backbones: a stable core composition and arrangement of genes in addition to variable regions. There are a few reports of extremely conserved plasmids. Here we report the complete sequence of pRK100 plasmid - a large, well-characterized conjugative F-like plasmid found in an Escherichia coli strain isolated from a urinary tract infection patient in 1990. The sequence shows that the 142 kb-long pRK100 plasmid is nearly identical to plasmids circulating in distant geographical locations and found in different host E. coli strains between 2007 and 2017. We also performed additional functional characterization of pRK100. Our results showed that pRK100 does not have a strong pathogenicity phenotype in porcine primary bladder epithelial cell culture. Moreover, the conjugation of pRK100 seems to strongly depend on recipient characteristics. These observations and identification of the pRK100 plasmid in different strain genotypes leave the extreme sequence conservation and broad distribution of this plasmid unexplained.

Molecular and Cell Biological Analysis of SwrB in Bacillus subtilis.

Swarming motility is flagellum-mediated movement over a solid surface, and Bacillus subtilis cells require an increase in flagellar density to swarm. SwrB is a protein of unknown function required for swarming that is necessary to increase the number of flagellar hooks but not basal bodies. Previous work suggested that SwrB activates flagellar type III secretion, but the mechanism by which it might perform this function is unknown. Here, we show that SwrB likely acts substoichiometrically as it localizes as puncta at the membrane in numbers fewer than those of flagellar basal bodies. Moreover, the action of SwrB is likely transient as puncta of SwrB were not dependent on the presence of the basal bodies and rarely colocalized with flagellar hooks. Random mutagenesis of the SwrB sequence found that a histidine within the transmembrane segment was conditionally required for activity and punctate localization. Finally, three hydrophobic residues that precede a cytoplasmic domain of poor conservation abolished SwrB activity when mutated and caused aberrant migration during electrophoresis. Our data are consistent with a model in which SwrB interacts with the flagellum, changes conformation to activate type III secretion, and departs. IMPORTANCE Type III secretion systems (T3SSs) are elaborate nanomachines that form the core of the bacterial flagellum and injectisome of pathogens. The machines not only secrete proteins like virulence factors but also secrete the structural components for their own assembly. Moreover, proper construction requires complex regulation to ensure that the parts are roughly secreted in the order in which they are assembled. Here, we explore a poorly understood activator of the flagellar T3SS activation in Bacillus subtilis called SwrB. To aid mechanistic understanding, we determine the rules for subcellular punctate localization, the topology with respect to the membrane, and critical residues required for SwrB function.

Benchmarking DNA isolation kits used in analyses of the urinary microbiome.

The urinary microbiome has been increasingly characterized using next-generation sequencing. However, many of the technical methods have not yet been specifically optimized for urine. We sought to compare the performance of several DNA isolation kits used in urinary microbiome studies. A total of 11 voided urine samples and one buffer control were divided into 5 equal aliquots and processed in parallel using five commercial DNA isolation kits. DNA was quantified and the V4 segment of the 16S rRNA gene was sequenced. Data were processed to identify the microbial composition and to assess alpha and beta diversity of the samples. Tested DNA isolation kits result in significantly different DNA yields from urine samples. DNA extracted with the Qiagen Biostic Bacteremia and DNeasy Blood & Tissue kits showed the fewest technical issues in downstream analyses, with the DNeasy Blood & Tissue kit also demonstrating the highest DNA yield. Nevertheless, all five kits provided good quality DNA for high throughput sequencing with non-significant differences in the number of reads recovered, alpha, or beta diversity.

Universal antibiotic tolerance arising from antibiotic-triggered accumulation of pyocyanin in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen that often infects open wounds or patients with cystic fibrosis. Once established, P. aeruginosa infections are notoriously difficult to eradicate. This difficulty is in part due to the ability of P. aeruginosa to tolerate antibiotic treatment at the individual-cell level or through collective behaviors. Here, we describe a new phenomenon by which P. aeruginosa tolerates antibiotic treatment. In particular, treatment of P. aeruginosa with sublethal concentrations of antibiotics covering all major classes promoted accumulation of the redox-sensitive phenazine pyocyanin (PYO). PYO in turn conferred general tolerance against diverse antibiotics for both P. aeruginosa and other gram-negative and gram-positive bacteria. This property is shared by other redox-active phenazines produced by P. aeruginosa. Our discovery sheds new insights into the physiological functions of phenazines and has implications for designing effective antibiotic treatment protocols.

Versatile biomanufacturing through stimulus-responsive cell-material feedback.

Small-scale production of biologics has great potential for enhancing the accessibility of biomanufacturing. By exploiting cell-material feedback, we have designed a concise platform to achieve versatile production, analysis and purification of diverse proteins and protein complexes. The core of our technology is a microbial swarmbot, which consists of a stimulus-sensitive polymeric microcapsule encapsulating engineered bacteria. By sensing the confinement, the bacteria undergo programmed partial lysis at a high local density. Conversely, the encapsulating material shrinks responding to the changing chemical environment caused by cell growth, squeezing out the protein products released by bacterial lysis. This platform is then integrated with downstream modules to enable quantification of enzymatic kinetics, purification of diverse proteins, quantitative control of protein interactions and assembly of functional protein complexes and multienzyme metabolic pathways. Our work demonstrates the use of the cell-material feedback to engineer a modular and flexible platform with sophisticated yet well-defined programmed functions.

Assessing heterogeneity in oligomeric AAA+ machines.

ATPases Associated with various cellular Activities (AAA+ ATPases) are molecular motors that use the energy of ATP binding and hydrolysis to remodel their target macromolecules. The majority of these ATPases form ring-shaped hexamers in which the active sites are located at the interfaces between neighboring subunits. Structural changes initiate in an active site and propagate to distant motor parts that interface and reshape the target macromolecules, thereby performing mechanical work. During the functioning cycle, the AAA+ motor transits through multiple distinct states. Ring architecture and placement of the catalytic sites at the intersubunit interfaces allow for a unique level of coordination among subunits of the motor. This in turn results in conformational differences among subunits and overall asymmetry of the motor ring as it functions. To date, a large amount of structural information has been gathered for different AAA+ motors, but even for the most characterized of them only a few structural states are known and the full mechanistic cycle cannot be yet reconstructed. Therefore, the first part of this work will provide a broad overview of what arrangements of AAA+ subunits have been structurally observed focusing on diversity of ATPase oligomeric ensembles and heterogeneity within the ensembles. The second part of this review will concentrate on methods that assess structural and functional heterogeneity among subunits of AAA+ motors, thus bringing us closer to understanding the mechanism of these fascinating molecular motors.

Dissecting the effects of antibiotics on horizontal gene transfer: Analysis suggests a critical role of selection dynamics.

Horizontal gene transfer (HGT) is a major mechanism responsible for the spread of antibiotic resistance. Conversely, it is often assumed that antibiotics promote HGT. Careful dissection of the literature, however, suggests a lack of conclusive evidence supporting this notion in general. This is largely due to the lack of well-defined quantitative experiments to address this question in an unambiguous manner. In this review, we discuss the extent to which HGT is responsible for the spread of antibiotic resistance and examine what is known about the effect of antibiotics on the HGT dynamics. We focus on conjugation, which is the dominant mode of HGT responsible for spreading antibiotic resistance on the global scale. Our analysis reveals a need to design experiments to quantify HGT in such a way to facilitate rigorous data interpretation. Such measurements are critical for developing novel strategies to combat resistance spread through HGT.