Survey of the infant male urobiome and genomic analysis of Actinotignum spp.

The urinary bladder harbors a community of microbes termed the urobiome, which remains understudied. In this study, we present the urobiome of healthy infant males from samples collected by transurethral catheterization. Using a combination of enhanced culture and amplicon sequencing, we identify several common bacterial genera that can be further investigated for their effects on urinary health across the lifespan. Many genera were shared between all samples suggesting a consistent urobiome composition among this cohort. We note that, for this cohort, early life exposures including mode of birth (vaginal vs. Cesarean section), or prior antibiotic exposure did not influence urobiome composition. In addition, we report the isolation of culturable bacteria from the bladders of these infant males, including Actinotignum spp., a bacterial genus that has been associated with urinary tract infections in older male adults. Herein, we isolate and sequence 9 distinct strains of Actinotignum spp. enhancing the genomic knowledge surrounding this genus and opening avenues for delineating the microbiology of this urobiome constituent. Furthermore, we present a framework for using the combination of culture-dependent and sequencing methodologies for uncovering mechanisms in the urobiome.

Defining the Infant Male Urobiome and Moving Towards Mechanisms in Urobiome Research.

The urinary bladder harbors a community of microbes termed the urobiome, which remains understudied. In this study, we present the urobiome of healthy infant males from samples collected by transurethral catheterization. Using a combination of extended culture and amplicon sequencing, we identify several common bacterial genera that can be further investigated for their effects on urinary health across the lifespan. Many genera were shared between all samples suggesting a consistent urobiome composition among this cohort. We note that, for this cohort, early life exposures including mode of birth (vaginal vs. Caesarean section), or prior antibiotic exposure did not influence urobiome composition. In addition, we report the isolation of culturable bacteria from the bladders of these infant males, including Actinotignum schaalii, a bacterial species that has been associated with urinary tract infection in older male adults. Herein, we isolate and sequence 9 distinct strains of A. schaalii enhancing the genomic knowledge surrounding this species and opening avenues for delineating the microbiology of this urobiome constituent. Furthermore, we present a framework for using the combination of culture-dependent and sequencing methodologies for uncovering mechanisms in the urobiome.

Urinary tract infections in cystic fibrosis patients.

Improved understanding of non-respiratory infections in cystic fibrosis (CF) patients will be vital to sustaining the increased life span of these patients. To date, there has not been a published report of urinary tract infections (UTIs) in CF patients. We performed a retrospective chart review at a major academic medical center during 2010-2020 to determine the features of UTIs in 826 CF patients. We identified 108 UTI episodes during this period. Diabetes, distal intestinal obstruction syndrome (DIOS), and nephrolithiasis were correlated with increased risk of UTIs. UTIs in CF patients were less likely to be caused by Gram-negative rods compared to non-CF patients and more likely to be caused by Enterococcus faecalis. The unique features of UTIs in CF patients highlight the importance of investigating non-respiratory infections to ensure appropriate treatment.

Cytochrome bd promotes Escherichia coli biofilm antibiotic tolerance by regulating accumulation of noxious chemicals.

Nutrient gradients in biofilms cause bacteria to organize into metabolically versatile communities capable of withstanding threats from external agents including bacteriophages, phagocytes, and antibiotics. We previously determined that oxygen availability spatially organizes respiration in uropathogenic Escherichia coli biofilms, and that the high-affinity respiratory quinol oxidase cytochrome bd is necessary for extracellular matrix production and biofilm development. In this study we investigate the physiologic consequences of cytochrome bd deficiency in biofilms and determine that loss of cytochrome bd induces a biofilm-specific increase in expression of general diffusion porins, leading to elevated outer membrane permeability. In addition, loss of cytochrome bd impedes the proton mediated efflux of noxious chemicals by diminishing respiratory flux. As a result, loss of cytochrome bd enhances cellular accumulation of noxious chemicals and increases biofilm susceptibility to antibiotics. These results identify an undescribed link between E. coli biofilm respiration and stress tolerance, while suggesting the possibility of inhibiting cytochrome bd as an antibiofilm therapeutic approach.

Data highlighting phenotypic diversity of urine-associated Escherichia coli isolates.

This article provides a reusable dataset describing detailed phenotypic and associated clinical parameters in n=303 clinical isolates of urinary Escherichia coli collected at Vanderbilt University Medical Center. De-identified clinical data collected with each isolate are detailed here and correlated to biofilm abundance and metabolomics data. Biofilm-abundance data were collected for each isolate under different in vitro conditions along with datasets quantifying biofilm abundance of each isolate under different conditions. Metabolomics data were collected from a subset of bacterial strains isolated from uncomplicated cases of cystitis or cases with no apparent symptoms accompanying colonization. For more insight, please see "Defining a Molecular Signature for Uropathogenic versus Urocolonizing Escherichia coli: The Status of the Field and New Clinical Opportunities" [1].

Defining a Molecular Signature for Uropathogenic versus Urocolonizing Escherichia coli: The Status of the Field and New Clinical Opportunities.

Urinary tract infections (UTIs) represent a major burden across the population, although key facets of their pathophysiology and host interaction remain unclear. Escherichia coli epitomizes these obstacles: this gram-negative bacterial species is the most prevalent agent of UTIs worldwide and can also colonize the urogenital tract in a phenomenon known as asymptomatic bacteriuria (ASB). Unfortunately, at the level of the individual E. coli strains, the relationship between UTI and ASB is poorly defined, confounding our understanding of microbial pathogenesis and strategies for clinical management. Unlike diarrheagenic pathotypes of E. coli, the definition of uropathogenic E. coli (UPEC) remains phenomenologic, without conserved phenotypes and known genetic determinants that rigorously distinguish UTI- and ASB-associated strains. This article provides a cross-disciplinary review of the current issues from interrelated mechanistic and diagnostic perspectives and describes new opportunities by which clinical resources can be leveraged to overcome molecular challenges. Specifically, we present our work harnessing a large collection of patient-derived isolates to identify features that do (and do not) distinguish UTI- from ASB-associated E. coli strains. Analyses of biofilm formation, previously reported to be higher in ASB strains, revealed extensive phenotypic heterogeneity that did not correlate with symptomatology. However, metabolomic experiments revealed distinct signatures between ASB and cystitis isolates, including in the purine pathway (previously shown to be critical for intracellular survival during acute infection). Together, these studies demonstrate how large-scale, wild-type approaches can help dissect the physiology of colonization versus infection, suggesting that the molecular definition of UPEC may rest at the level of global bacterial metabolism.

Using Multiplex Molecular Testing to Determine the Etiology of Acute Gastroenteritis in Children.

OBJECTIVE: To detect the etiologic agents of acute gastroenteritis (AGE) in children using broad molecular-based techniques, and compare clinical presentations among etiologies. STUDY DESIGN: This was a prospective population-based surveillance study of children aged <6 years with AGE conducted between 2008 and 2011 as part of the New Vaccine Surveillance Network. Stools from patients and healthy controls were tested for 21 gastrointestinal pathogens using the analyte-specific reagent Gastrointestinal Pathogen Panel and an additional reverse transcription real-time polymerase chain reaction assay for sapovirus and astrovirus. RESULTS: Of the 216 stool samples from patients with AGE, 152 (70.4%) tested positive for a pathogen, with norovirus genogroup II (n = 78; 36.1%) and Clostridium difficile (n = 35; 16.2%) the most common pathogens detected. Forty-nine patients (22.7%) tested positive for more than 1 pathogen, including 25 (71%) with a C difficile detection. There were no significant clinical differences among the patients with no pathogen detected, those with a single pathogen detected, and those with ≥2 pathogens detected. CONCLUSION: Using a broad molecular testing approach, high rates of enteropathogens were detected in children with AGE, dominated by norovirus genogroup II and C difficile. Coinfections were common but had no identifiable impact on clinical manifestations. As routine diagnostics of AGE progressively evolve toward nucleic acid-based pathogen detection, ongoing systematic studies are needed to better analyze the clinical significance of results.

Coccidioides, cryptococcus, or blastomyces? A diagnostic dilemma encountered during frozen section evaluation.

Intraoperative consultation via frozen section is an important part of modern day surgical pathology. Recognizing fungi in tissues on frozen and permanent sections is not always a simple task, and correctly identifying the agent can be a significant challenge, even for experienced microscopists. We present a case of a 17-year-old boy with chronic osteomyelitis involving the right proximal ulna. During an irrigation and debridement operation, a frozen section was sent to surgical pathology for evaluation. A limited patient history coupled with sparse organisms present in the frozen section led to the diagnosis of fungal osteomyelitis, favor Coccidioides . Follow-up permanent sections with special staining and successful fungal culture clarified the causal agent to be Blastomyces dermatitidis . The role of frozen sections is not to perfectly speciate the fungal pathogen but to describe the morphology and infectious process and provide a differential diagnosis of the candidate fungi. The importance of intraoperative culture in infectious cases cannot be understated, and it is the responsibility of pathologists to inform surgeons that tissue is needed for culture. A brief overview of Blastomyces , including histopathologic features and key microscopic differences from Coccidioides and Cryptococcus , is discussed.

Purification and biochemical characterization of the E1 replication initiation protein of the cutaneous human papillomavirus type 1.

The E1 and E2 proteins encoded by papillomaviruses are required for viral DNA replication. Although E1 is the replication initiator protein, previous studies have shown that the full-length E1 protein binds to the origin weakly and with low sequence specificity. The E2 protein facilitates binding of the E1 protein to the origin, triggering the initiation of replication. The E1 protein contains ATPase, helicase and DNA unwinding activities. In vivo studies with mucosal human papillomavirus (HPV) types 11 and 18 have shown that while E1 is absolutely essential for replication, the E1 binding site is dispensable. However, both the E2 protein and E2 binding sites are required for their replication. In contrast to these HPVs, transient replication of HPV type 1, which infects cutaneous tissue, requires only the viral E1 protein and E1 binding site. To understand the basis for these differences, we have overexpressed and purified the HPV-1 E1 and E2 proteins and studied their biochemical properties. The purified E1 protein was shown to have an ATPase activity with a very low K(m) value, similar to that of the SV40 large T antigen. The E1 protein bound to the HPV-1 origin in the absence of the E2 protein and without the use of any cross-linking agents. Our results suggest that the ability of the HPV-1 E1 protein to initiate DNA replication in vivo in the absence of the E2 protein may be due to its stable interaction with the HPV-1 origin.