Evelyn Toh

Evidence of Uncultivated Bacteria in the Adult Female Bladder

ABSTRACT Clinical urine specimens are usually considered to be sterile when they do not yield uropathogens using standard clinical cultivation procedures. Our aim was to test if the adult female bladder might contain bacteria that are not identified by these routine procedures. An additional aim was to identify and recommend the appropriate urine collection method for the study of bacterial communities in the female bladder. Consenting participants who were free of known urinary tract infection provided urine samples by voided, transurethral, and/or suprapubic collection methods. The presence of bacteria in these samples was assessed by bacterial culture, light microscopy, and 16S rRNA gene sequencing. Bacteria that are not or cannot be routinely cultivated (hereinafter called uncultivated bacteria) were common in voided urine, urine collected by transurethral catheter (TUC), and urine collected by suprapubic aspirate (SPA), regardless of whether the subjects had urinary symptoms. Voided urine samples contained mixtures of urinary and genital tract bacteria. Communities identified in parallel urine samples collected by TUC and SPA were similar. Uncultivated bacteria are clearly present in the bladders of some women. It remains unclear if these bacteria are viable and/or if their presence is relevant to idiopathic urinary tract conditions.

The Microbial Communities in Male First Catch Urine Are Highly Similar to Those in Paired Urethral Swab Specimens

Urine is the CDC-recommended specimen for STI testing. It was unknown if the bacterial communities (microbiomes) in urine reflected those in the distal male urethra. We compared microbiomes of 32 paired urine and urethral swab specimens obtained from adult men attending an STD clinic, by 16S rRNA PCR and deep pyrosequencing. Microbiomes of urine and swabs were remarkably similar, regardless of STI status of the subjects. Thus, urine can be used to characterize urethral microbiomes when swabs are undesirable, such as in population-based studies of the urethral microbiome or where multiple sampling of participants is required.

Bacterial Communities of the Coronal Sulcus and Distal Urethra of Adolescent Males

Lactobacillus-dominated vaginal microbiotas are associated with reproductive health and STI resistance in women, whereas altered microbiotas are associated with bacterial vaginosis (BV), STI risk and poor reproductive outcomes. Putative vaginal taxa have been observed in male first-catch urine, urethral swab and coronal sulcus (CS) specimens but the significance of these observations is unclear. We used 16 S rRNA sequencing to characterize the microbiota of the CS and urine collected from 18 adolescent men over three consecutive months. CS microbiotas of most participants were more stable than their urine microbiotas and the composition of CS microbiotas were strongly influenced by circumcision. BV-associated taxa, including Atopobium, Megasphaera, Mobiluncus, Prevotella and Gemella, were detected in CS specimens from sexually experienced and inexperienced participants. In contrast, urine primarily contained taxa that were not abundant in CS specimens. Lactobacilllus and Streptococcus were major urine taxa but their abundance was inversely correlated. In contrast, Sneathia, Mycoplasma and Ureaplasma were only found in urine from sexually active participants. Thus, the CS and urine support stable and distinct bacterial communities. Finally, our results suggest that the penis and the urethra can be colonized by a variety of BV-associated taxa and that some of these colonizations result from partnered sexual activity.

Development of Surface Adhesion in Caulobacter crescentus

Caulobacter crescentus has a dimorphic life cycle composed of a motile stage and a sessile stage. In the sessile stage, C. crescentus is often found tightly attached to a surface through its adhesive holdfast. In this study, we examined the contribution of growth and external structures to the attachment of C. crescentus to abiotic surfaces. We show that the holdfast is essential but not sufficient for optimal attachment. Rather, adhesion in C. crescentus is a complex developmental process. We found that the attachment of C. crescentus to surfaces is cell cycle regulated and that growth or energy or both are essential for this process. The initial stage of attachment occurs in swarmer cells and is facilitated by flagellar motility and pili. Our results suggest that strong attachment is mediated by the synthesis of a holdfast as the swarmer cell differentiates into a stalked cell.

The arthropod, but not the vertebrate host or its environment, dictates bacterial community composition of fleas and ticks

Bacterial community composition in blood-sucking arthropods can shift dramatically across time and space. We used 16S rRNA gene amplification and pyrosequencing to investigate the relative impact of vertebrate host-related, arthropod-related and environmental factors on bacterial community composition in fleas and ticks collected from rodents in southern Indiana (USA). Bacterial community composition was largely affected by arthropod identity, but not by the rodent host or environmental conditions. Specifically, the arthropod group (fleas vs ticks) determined the community composition of bacteria, where bacterial communities of ticks were less diverse and more dependent on arthropod traits—especially tick species and life stage—than bacterial communities of fleas. Our data suggest that both arthropod life histories and the presence of arthropod-specific endosymbionts may mask the effects of the vertebrate host and its environment.

Characterization of the Caulobacter crescentus Holdfast Polysaccharide Biosynthesis Pathway Reveals Significant Redundancy in the Initiating Glycosyltransferase and Polymerase Steps

Caulobacter crescentus cells adhere to surfaces by using an extremely strong polar adhesin called the holdfast. The polysaccharide component of the holdfast is comprised in part of oligomers of N-acetylglucosamine. The genes involved in the export of the holdfast polysaccharide and the anchoring of the holdfast to the cell were previously discovered. In this study, we identified a cluster of polysaccharide biosynthesis genes (hfsEFGH) directly adjacent to the holdfast polysaccharide export genes. Sequence analysis indicated that these genes are involved in the biosynthesis of the minimum repeat unit of the holdfast polysaccharide. HfsE is predicted to be a UDP-sugar lipid-carrier transferase, the glycosyltransferase that catalyzes the first step in polysaccharide biosynthesis. HfsF is predicted to be a flippase, HfsG is a glycosyltransferase, and HfsH is similar to a polysaccharide (chitin) deacetylase. In-frame hfsG and hfsH deletion mutants resulted in severe deficiencies both in surface adhesion and in binding to the holdfast-specific lectin wheat germ agglutinin. In contrast, hfsE and hfsF mutants exhibited nearly wild-type levels of adhesion and holdfast synthesis. We identified three paralogs to hfsE, two of which are redundant to hfsE for holdfast synthesis. We also identified a redundant paralog to the hfsC gene, encoding the putative polysaccharide polymerase, and present evidence that the hfsE and hfsC paralogs, together with the hfs genes, are absolutely required for proper holdfast synthesis.

A localized multimeric anchor attaches the Caulobacter holdfast to the cell pole

Caulobacter crescentus attachment is mediated by the holdfast, a complex of polysaccharide anchored to the cell by HfaA, HfaB and HfaD. We show that all three proteins are surface exposed outer membrane (OM) proteins. HfaA is similar to fimbrial proteins and assembles into a high molecular weight (HMW) form requiring HfaD, but not holdfast polysaccharide. The HfaD HMW form is dependent on HfaA but not on holdfast polysaccharide. We show that HfaA and HfaD form homomultimers and that they require HfaB for stability and OM translocation. All three proteins localize to the late pre‐divisional flagellar pole, remain at this pole in swarmer cells, and localize at the stalk tip after the stalk is synthesized at the same pole. Hfa protein localization requires the holdfast polysaccharide secretion proteins and the polar localization factor PodJ. An hfaB mutant is much more severely deficient in adherence and holdfast attachment than hfaA and hfaD mutants. An hfaA, hfaD double mutant phenocopies either single mutant, suggesting that HfaB is involved in holdfast attachment beyond secretion of HfaA and HfaD. We hypothesize that HfaB secretes HfaA and HfaD across the outer membrane, and the three proteins form a complex anchoring the holdfast to the stalk.

Interplay between Bladder Microbiota and Urinary Antimicrobial Peptides: Mechanisms for Human Urinary Tract Infection Risk and Symptom Severity

Resident bacterial communities (microbiota) and host antimicrobial peptides (AMPs) are both essential components of normal host innate immune responses that limit infection and pathogen induced inflammation. However, their interdependence has not been investigated in the context of urinary tract infection (UTI) susceptibility. Here, we explored the interrelationship between the urinary microbiota and host AMP responses as mechanisms for UTI risk. Using prospectively collected day of surgery (DOS) urine specimens from female pelvic floor surgery participants, we report that the relative abundance and/or frequency of specific urinary microbiota distinguished between participants who did or did not develop a post-operative UTI. Furthermore, UTI risk significantly correlated with both specific urinary microbiota and β-defensin AMP levels. Finally, urinary AMP hydrophobicity and protease activity were greater in participants who developed UTI, and correlated positively with both UTI risk and pelvic floor symptoms. These data demonstrate an interdependency between the urinary microbiota, AMP responses and symptoms, and identify a potential mechanism for UTI risk. Assessment of bacterial microbiota and host innate immune AMP responses in parallel may identify increased risk of UTI in certain populations.

The Human Skin Microbiome Associates with the Outcome of and Is Influenced by Bacterial Infection

ABSTRACT The influence of the skin microbiota on host susceptibility to infectious agents is largely unexplored. The skin harbors diverse bacterial species that may promote or antagonize the growth of an invading pathogen. We developed a human infection model for Haemophilus ducreyi in which human volunteers are inoculated on the upper arm. After inoculation, papules form and either spontaneously resolve or progress to pustules. To examine the role of the skin microbiota in the outcome of H. ducreyi infection, we analyzed the microbiomes of four dose-matched pairs of “resolvers” and “pustule formers” whose inoculation sites were swabbed at multiple time points. Bacteria present on the skin were identified by amplification and pyrosequencing of 16S rRNA genes. Nonmetric multidimensional scaling (NMDS) using Bray-Curtis dissimilarity between the preinfection microbiomes of infected sites showed that sites from the same volunteer clustered together and that pustule formers segregated from resolvers (P = 0.001, permutational multivariate analysis of variance [PERMANOVA]), suggesting that the preinfection microbiomes were associated with outcome. NMDS using Bray-Curtis dissimilarity of the endpoint samples showed that the pustule sites clustered together and were significantly different than the resolved sites (P = 0.001, PERMANOVA), suggesting that the microbiomes at the endpoint differed between the two groups. In addition to H. ducreyi, pustule-forming sites had a greater abundance of Proteobacteria, Bacteroidetes, Micrococcus, Corynebacterium, Paracoccus, and Staphylococcus species, whereas resolved sites had higher levels of Actinobacteria and Propionibacterium species. These results suggest that at baseline, resolvers and pustule formers have distinct skin bacterial communities which change in response to infection and the resultant immune response. IMPORTANCE Human skin is home to a diverse community of microorganisms, collectively known as the skin microbiome. Some resident bacteria are thought to protect the skin from infection by outcompeting pathogens for resources or by priming the immune systems response to invaders. However, the influence of the skin microbiome on the susceptibility to or protection from infection has not been prospectively evaluated in humans. We characterized the skin microbiome before, during, and after experimental inoculation of the arm with Haemophilus ducreyi in matched volunteers who subsequently resolved the infection or formed abscesses. Our results suggest that the preinfection microbiomes of pustule formers and resolvers have distinct community structures which change in response to the progression of H. ducreyi infection to abscess formation. Human skin is home to a diverse community of microorganisms, collectively known as the skin microbiome. Some resident bacteria are thought to protect the skin from infection by outcompeting pathogens for resources or by priming the immune systems response to invaders. However, the influence of the skin microbiome on the susceptibility to or protection from infection has not been prospectively evaluated in humans. We characterized the skin microbiome before, during, and after experimental inoculation of the arm with Haemophilus ducreyi in matched volunteers who subsequently resolved the infection or formed abscesses. Our results suggest that the preinfection microbiomes of pustule formers and resolvers have distinct community structures which change in response to the progression of H. ducreyi infection to abscess formation.

The HfaB and HfaD adhesion proteins of Caulobacter crescentus are localized in the stalk

The differentiating bacterium Caulobacter crescentus produces two different cell types at each cell division, a motile swarmer cell and an adhesive stalked cell. The stalked cell harbours a stalk, a thin cylindrical extension of the cell surface. The tip of the stalk is decorated with a holdfast, an adhesive organelle composed at least in part of polysaccharides. The synthesis of the stalk and holdfast occur at the same pole during swarmer cell differentiation. Mutations in the hfaABDC gene cluster had been shown to disrupt the attachment of the holdfast to the tip of the stalk, but the role of individual genes was unknown. We used lacZ fusions of various DNA fragments from the hfaABDC region to show that these genes form an operon. In order to analyse the relative contribution of the different genes to holdfast attachment, mutations were constructed for each gene. hfaC was not required for holdfast attachment or binding to surfaces. The hfaA and hfaD mutants shed some holdfast material into the surrounding medium and were partially deficient in binding to surfaces. Unlike hfaA and hfaB mutants, hfaD mutants were still able to form rosettes efficiently. Cells with insertions in hfaB were unable to bind to surfaces, and lectin binding studies indicated that the hfaB mutants had the strongest holdfast shedding phenotype. We determined that HfaB and HfaD are membrane‐associated proteins and that HfaB is a lipoprotein. Purification of stalks and cell bodies indicated that both HfaB and HfaD are enriched in the stalk as compared to the cell body. These results suggest that HfaB and HfaD, and probably HfaA, serve to anchor the holdfast to the tip of the stalk.