Raising the alarm: fosfomycin resistance associated with non-susceptible inner colonies imparts no fitness cost to the primary bacterial uropathogen.

IMPORTANCE: While fosfomycin resistance is rare, the observation of non-susceptible subpopulations among clinical Escherichia coli isolates is a common phenomenon during antimicrobial susceptibility testing (AST) in American and European clinical labs. Previous evidence suggests that mutations eliciting this phenotype are of high biological cost to the pathogen during infection, leading to current recommendations of neglecting non-susceptible colonies during AST. Here, we report that the most common route to fosfomycin resistance, as well as novel routes described in this work, does not impair virulence in uropathogenic E. coli, the major cause of urinary tract infections, suggesting a re-evaluation of current susceptibility guidelines is warranted.

Machine-Learning Model for Prediction of Cefepime Susceptibility in Escherichia coli from Whole-Genome Sequencing Data.

The declining cost of performing bacterial whole-genome sequencing (WGS) coupled with the availability of large libraries of sequence data for well-characterized isolates have enabled the application of machine-learning (ML) methods to the development of nonlinear sequence-based predictive models. We tested the ML-based model developed by Next Gen Diagnostics for prediction of cefepime phenotypic susceptibility results in Escherichia coli. A cohort of 100 isolates of E. coli recovered from urine (n = 77) and blood (n = 23) cultures were used. The cefepime MIC was determined in triplicate by reference broth microdilution and classified as susceptible (MIC of ≤2 µg/mL) or not susceptible (MIC of ≥4 µg/mL) using the 2022 Clinical and Laboratory Standards Institute breakpoints. Five isolates generated both susceptible and not susceptible MIC results, yielding categorical agreement of 95% for the reference method to itself. Categorical agreement of ML to MIC interpretations was 97%, with 2 very major (false, susceptible) and 1 major (false, not susceptible) errors. One very major error occurred for an isolate with blaCTX-M-27 (MIC mode, ≥32 µg/mL) and one for an isolate with blaTEM-34 for which the MIC cefepime mode was 4 µg/mL. One major error was for an isolate with blaCTX-M-27 but with a MIC mode of 2 µg/mL. These preliminary data demonstrated performance of ML for a clinically important antimicrobial-species pair at a caliber similar to phenotypic methods, encouraging wider development of sequence-based susceptibility prediction and its validation and use in clinical practice.

Intra-strain colony biofilm heterogeneity in uropathogenic Escherichia coli and the effect of the NlpI lipoprotein.

Biofilm growth facilitates the interaction of uropathogenic Escherichia coli (UPEC) with the host environment. The extracellular polymeric substances (EPS) of UPEC biofilms are composed prominently of curli amyloid fiber and cellulose polysaccharide. When the organism is propagated as a colony biofilm on agar media, these macromolecules can generate pronounced macroscopic structures. Moreover, curli/cellulose associate tightly with Congo red, generating a characteristic pink-to-red staining pattern when the media is supplemented with this dye. Among different clinical isolates of UPEC, changes in the abundance of curli/cellulose can lead to diverse colony biofilm phenotypes on a strain-by-strain basis. Nevertheless, for any given isolate, these phenotypes are classically homogenous throughout the colony biofilm. Here, we report that a subset of clinical UPEC isolates display heterogenous 'peppermint' colony biofilms, with distinct pale and red subpopulations. Through isolation of these subpopulations and whole genome sequencing, we demonstrate various emergent mutations associated with the phenomenon, including within the gene encoding the outer membrane lipoprotein nlpI. Deletion of nlpI within independent strain-backgrounds increased biofilm rugosity, while its overexpression induced the peppermint phenotype. Upregulation of EPS-associated proteins and transcripts was likewise observed in the absence of nlpI. Overall, these results demonstrate that EPS elaboration in UPEC is impacted by nlpI. More broadly, this phenomenon of intra-strain colony biofilm heterogeneity may be leveraged as a tool to identify additional members within the broad collection of genes that regulate or otherwise affect biofilm formation.

The Role of Mobile Genetic Elements in Virulence Factor Carriage from Symptomatic and Asymptomatic Cases of Escherichia coli Bacteriuria.

Uropathogenic Escherichia coli (UPEC) is extremely diverse genotypically and phenotypically. Individual strains can variably carry diverse virulence factors, making it challenging to define a molecular signature for this pathotype. For many bacterial pathogens, mobile genetic elements (MGEs) constitute a major mechanism of virulence factor acquisition. For urinary E. coli, the total distribution of MGEs and their role in the acquisition of virulence factors is not well defined, including in the context of symptomatic infection versus asymptomatic bacteriuria (ASB). In this work, we characterized 151 isolates of E. coli, derived from patients with either urinary tract infection (UTI) or ASB. For both sets of E. coli, we catalogued the presence of plasmids, prophage, and transposons. We analyzed MGE sequences for the presence of virulence factors and antimicrobial resistance genes. These MGEs were associated with only ~4% of total virulence associated genes, while plasmids contributed to ~15% of antimicrobial resistance genes under consideration. Our analyses suggests that, across strains of E. coli, MGEs are not a prominent driver of urinary tract pathogenesis and symptomatic infection. IMPORTANCE Escherichia coli is the most common etiological agent of urinary tract infections (UTIs), with UTI-associated strains designated "uropathogenic" E. coli or UPEC. Across urinary strains of E. coli, the global landscape of MGEs and its relationship to virulence factor carriage and clinical symptomatology require greater clarity. Here, we demonstrate that many of the putative virulence factors of UPEC are not associated with acquisition due to MGEs. The current work enhances our understanding of the strain-to-strain variability and pathogenic potential of urine-associated E. coli and points toward more subtle genomic differences distinguishing ASB from UTI isolates.

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.

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.

Uropathogenic Escherichia coli subverts mitochondrial metabolism to enable intracellular bacterial pathogenesis in urinary tract infection.

Urinary tract infections are among the most common human bacterial infections and place a significant burden on healthcare systems due to associated morbidity, cost and antibiotic use. Despite being a facultative anaerobe, uropathogenic Escherichia coli, the primary cause of urinary tract infections, requires aerobic respiration to establish infection in the bladder. Here, by combining bacterial genetics with cell culture and murine models of infection, we demonstrate that the widely conserved respiratory quinol oxidase cytochrome bd is required for intracellular infection of urothelial cells. Through a series of genetic, biochemical and functional assays, we show that intracellular oxygen scavenging by cytochrome bd alters mitochondrial physiology by reducing the efficiency of mitochondrial respiration, stabilizing the hypoxia-inducible transcription factor HIF-1 and promoting a shift towards aerobic glycolysis. This bacterially induced rewiring of host metabolism antagonizes apoptosis, thereby protecting intracellular bacteria from urothelial cell exfoliation and preserving their replicative niche. These results reveal the metabolic basis for intracellular bacterial pathogenesis during urinary tract infection and identify subversion of mitochondrial metabolism as a bacterial strategy to facilitate persistence within the urinary tract.

BioKIT: a versatile toolkit for processing and analyzing diverse types of sequence data.

Bioinformatic analysis-such as genome assembly quality assessment, alignment summary statistics, relative synonymous codon usage, file format conversion, and processing and analysis-is integrated into diverse disciplines in the biological sciences. Several command-line pieces of software have been developed to conduct some of these individual analyses, but unified toolkits that conduct all these analyses are lacking. To address this gap, we introduce BioKIT, a versatile command line toolkit that has, upon publication, 42 functions, several of which were community-sourced, that conduct routine and novel processing and analysis of genome assemblies, multiple sequence alignments, coding sequences, sequencing data, and more. To demonstrate the utility of BioKIT, we conducted a comprehensive examination of relative synonymous codon usage across 171 fungal genomes that use alternative genetic codes, showed that the novel metric of gene-wise relative synonymous codon usage can accurately estimate gene-wise codon optimization, evaluated the quality and characteristics of 901 eukaryotic genome assemblies, and calculated alignment summary statistics for 10 phylogenomic data matrices. BioKIT will be helpful in facilitating and streamlining sequence analysis workflows. BioKIT is freely available under the MIT license from GitHub (https://github.com/JLSteenwyk/BioKIT), PyPi (https://pypi.org/project/jlsteenwyk-biokit/), and the Anaconda Cloud (https://anaconda.org/jlsteenwyk/jlsteenwyk-biokit). Documentation, user tutorials, and instructions for requesting new features are available online (https://jlsteenwyk.com/BioKIT).

Redox-Mediated Inactivation of the Transcriptional Repressor RcrR is Responsible for Uropathogenic Escherichia coli's Increased Resistance to Reactive Chlorine Species.

The ability to overcome stressful environments is critical for pathogen survival in the host. One challenge for bacteria is the exposure to reactive chlorine species (RCS), which are generated by innate immune cells as a critical part of the oxidative burst. Hypochlorous acid (HOCl) is the most potent antimicrobial RCS and is associated with extensive macromolecular damage in the phagocytized pathogen. However, bacteria have evolved defense strategies to alleviate the effects of HOCl-mediated damage. Among these are RCS-sensing transcriptional regulators that control the expression of HOCl-protective genes under non-stress and HOCl stress. Uropathogenic Escherichia coli (UPEC), the major causative agent of urinary tract infections (UTIs), is particularly exposed to infiltrating neutrophils during pathogenesis; however, their responses to and defenses from HOCl are still completely unexplored. Here, we present evidence that UPEC strains tolerate higher levels of HOCl and are better protected from neutrophil-mediated killing compared with other E. coli. Transcriptomic analysis of HOCl-stressed UPEC revealed the upregulation of an operon consisting of three genes, one of which encodes the transcriptional regulator RcrR. We identified RcrR as a HOCl-responsive transcriptional repressor, which, under non-stress conditions, is bound to the operator and represses the expression of its target genes. During HOCl exposure, however, the repressor forms reversible intermolecular disulfide bonds and dissociates from the DNA resulting in the derepression of the operon. Deletion of one of the target genes renders UPEC significantly more susceptible to HOCl and phagocytosis indicating that the HOCl-mediated induction of the regulon plays a major role for UPEC's HOCl resistance. IMPORTANCE How do pathogens deal with antimicrobial oxidants produced by the innate immune system during infection? Uropathogenic Escherichia coli (UPEC), the most common etiological agent of urinary tract infections (UTIs), is particularly exposed to infiltrating neutrophils and, therefore, must counter elevated levels of the antimicrobial oxidant HOCl to establish infection. Our study provides fundamentally new insights into a defense mechanism that enables UPEC to fend off the toxic effects of HOCl stress. Intriguingly, the defense system is predominantly found in UPEC and absent in noninvasive enteropathogenic E. coli. Our data suggest expression of the target gene rcrB is exclusively responsible for UPEC's increased HOCl tolerance in culture and contributes to UPEC's survival during phagocytosis. Thus, this novel HOCl stress defense system could potentially serve as an attractive drug target to increase the body's own capacity to fight UTIs.

Whole genome sequencing of Rhodotorula mucilaginosa isolated from the chewing stick (Distemonanthus benthamianus): insights into Rhodotorula phylogeny, mitogenome dynamics and carotenoid biosynthesis.

In industry, the yeast Rhodotorula mucilaginosa is commonly used for the production of carotenoids. The production of carotenoids is important because they are used as natural colorants in food and some carotenoids are precursors of retinol (vitamin A). However, the identification and molecular characterization of the carotenoid pathway/s in species belonging to the genus Rhodotorula is scarce due to the lack of genomic information thus potentially impeding effective metabolic engineering of these yeast strains for improved carotenoid production. In this study, we report the isolation, identification, characterization and the whole nuclear genome and mitogenome sequence of the endophyte R. mucilaginosa RIT389 isolated from Distemonanthus benthamianus, a plant known for its anti-fungal and antibacterial properties and commonly used as chewing sticks. The assembled genome of R. mucilaginosa RIT389 is 19 Mbp in length with an estimated genomic heterozygosity of 9.29%. Whole genome phylogeny supports the species designation of strain RIT389 within the genus in addition to supporting the monophyly of the currently sequenced Rhodotorula species. Further, we report for the first time, the recovery of the complete mitochondrial genome of R. mucilaginosa using the genome skimming approach. The assembled mitogenome is at least 7,000 bases larger than that of Rhodotorula taiwanensis which is largely attributed to the presence of large intronic regions containing open reading frames coding for homing endonuclease from the LAGLIDADG and GIY-YIG families. Furthermore, genomic regions containing the key genes for carotenoid production were identified in R. mucilaginosa RIT389, revealing differences in gene synteny that may play a role in the regulation of the biotechnologically important carotenoid synthesis pathways in yeasts.

Incidencia de infección respiratoria aguda en las comunidades de Cevallos, Yanahurco y Quinchicoto Tunguharua 1996-1997

Desde que en el año 1979 la comunidad internacional reconoció a las infecciones respiratorias agudas (IRA) como un grave problema de salud que afecta a la infancia, la región de las américas ha desarrollado un esfuerzo sistemático para combatir este problema, por lo que a emprendido programas de control tendientes a disminuir la mortalidad por IRA especialmente por Neumonía (9), ubicándose esta patología entre una de las primeras 5 causas de muerte infantil además de enfermedades Diarréicas, Desnutrición, Malaria y Sarampión (5,8), representando esta entidad alrededor del 70 por ciento de las defunciones infantiles en 88 países del planeta (5.1) y con tasas de mortalidad mayores de 40/1000 nacidos vivos. La importancia que el problema de las IRA tiene sobre la salud del niño, ha hecho que su control represente una prioridad para los países en vías de desarrollo especialmente con altas tasas de mortalidad. Los menores de 5 años son el grupo más propenso a sufrir estos transtornos y sus complicaciones. Según la OMS, este grupo contrae de 6 a 8 episodios anuales de IRA. Se ha descubierto que por cada episodio anual de infección de la vía respiratoria inferior hay de 4 a 14 episodios de la vía respiratoria superior.

Papanicolaou como medio diagnóstico de gérmenes asociados a cérvico-vaginitis en un grupo de mujeres de la Parroquia Atahualpa - Ambato 1997

El papanicolaou es un examen utilizado para la detección oportuna del cáncer cérvicouterino y a la vez ayuda a detectar los diferentes procesos inflamatorios e infecciosos del tracto genital inferior. El presente trabajo expone los resultados citológicos así como los gérmenes encontrados con mayor frecuencia en los frotis de un grupo de mujeres de la parroquia Atahualpa Ambato en el que el germen predominante fue la gardnerella vaginalis en un 29.87 por ciento del total de la muestra.

Hematoma subdural crónico: revisión de la bibliografía a propósito de un caso clínico / chronic subdural Hematoma: overhaul of the bibliography with regard to a clinical case

Presenta un caso de cefalea crónica progresiva en un paciente varón de 60 años de edad, acompañada de torpeza mental y sensación vertiginosa. El hematoma subdural crónico (HSC) es una colección de sangre venosa que se localiza por debajo de la duramadre, cuya etiología traumática es trivial; más frecuente en ancianos y alcohólicos. Se manifiesta clínicamente en un período mayor a 14 días, el diagnóstico debe ser inmediato por tomografía computarizada (TC) efectuando posteriormente evacuación quirúrgica.

Incidencia de infección respiratoria aguda en las comunidades de Cevallos, Yanahurco y Quinchicoto-Tungurahua 1996-1997

Se estudiaron retrospectivamente 644 casos de pacientes que acudieron a consulta externa del centro de salud del Cantón Cevallos, los subcentros Quinchicoto y del Caserio Yanahurco, en la provincia de Tungurahua, desde 1996-1997. A este grupo de pacientes se los dividió en 2 grupos de estudio. Menores de 1 año y de 1 a 4 años; encontrándose una mayor incidencia de IRA en el primer grupo con un 54,5 por ciento en relación a 45,5 por ciento encontrado en el 2do. grupo; donde se demuestra, que los problemas de tipo viral son los que tienen más incidencia (70,4 por ciento), y; los de tipo bacteriano con una menor incidencia en pacientes menores de 1 año (12 pctes=1,8 por ciento), y; en los pacientes de 1 a 4 años (3 pactes=0,4 por ciento); este estudio se complementa con una revisión sobre el tratamiento de elección.

Papanicolau como medio diagnòstico de gèrmenes asociados a cèrvico-vaginitis en un grupo de mujeres de la parroquia Atahualpa Ambato 1997

El papanicolau es un examen utilizado para la detecciòn oportuna del càncer cèrvicouterino y a la vez ayuda a detectar los diferentes procesos inflamatorios e infecciosos del tracto genital inferior. El presente trabajo expone los resultados citològicos asì como los gèrmenes encontrados con mayor frecuencia en los frotis de un grupo de mujeres de la parroquia Atahualpa Ambato en el que el germen predominante fue la gardnerella vaginalis en un 29.87 por ciento del total de la muestra.

Enfermedad diarréica aguda en las comunidades de Cevallos, Quinchicoto y Yanahurco provincia de Tungurahua 1996-1997 / acute diarreica Disease in the communities of Cevallos, Quinchicoto and Yanahurco province of Tungurahua 1996-1997

Presenta un estudio de carácter retrospectivo, analizando las historias clínicas de 96 pacientes que acudieron a la consulta externa del Centro de Salud de la parroquia de Quinchicoto y del Caserío de Yanahurco en la provincia de Tungurahua, desde el 01 de ene. 1996 hasta ju. 30 de 1997. Tomando en cuenta la edad, sexo, tiempo de duración del episodio de diarrea y el tratamiento que recibió. Se los dividió en dos grupos: El primero, de menores de 1 año donde la mayoría de pacientes son del sexo femenino (65 por ciento), con un tiempo de duración de la diarrea de 5,1 días promedio (rango de 1 a 8 días), el tratamiento empleado fue Plan A (según el MSP) en un 67,5 por ciento donde el antibiótico de elección fue Trimetoprin sulfametoxazol en un 20 por ciento de los pacientes. En el segundo grupo, de menores de 1 a 4 años, la mayoría pertenece al sexo femenino (55,3 por ciento), con un tiempo de duración de la diarrea de 4,7 días promedio (rango de 2 a 12 días), el tratamiento empleado fue Plan A en un 64,2 por ciento, y los antibióticos utilizados fueron Trimetoprin Sulfametoxazol en 10,7 por ciento. Se complementa el estudio con una revisión de los planes de tratamiento para EDA según el MSP-Ecuador.