RESUMEN
A disrupted "dysbiotic" gut microbiome engenders susceptibility to the diarrheal pathogen Clostridioides difficile by impacting the metabolic milieu of the gut. Diet, in particular the microbiota-accessible carbohydrates (MACs) found in dietary fiber, is one of the most powerful ways to affect the composition and metabolic output of the gut microbiome. As such, diet is a powerful tool for understanding the biology of C. difficile and for developing alternative approaches for coping with this pathogen. One prominent class of metabolites produced by the gut microbiome is short-chain fatty acids (SCFAs), the major metabolic end products of MAC metabolism. SCFAs are known to decrease the fitness of C. difficile in vitro, and high intestinal SCFA concentrations are associated with reduced fitness of C. difficile in animal models of C. difficile infection (CDI). Here, we use controlled dietary conditions (8 diets that differ only by MAC composition) to show that C. difficile fitness is most consistently impacted by butyrate, rather than the other two prominent SCFAs (acetate and propionate), during murine model CDI. We similarly show that butyrate concentrations are lower in fecal samples from humans with CDI than in those from healthy controls. Finally, we demonstrate that butyrate impacts growth in diverse C. difficile isolates. These findings provide a foundation for future work which will dissect how butyrate directly impacts C. difficile fitness and will lead to the development of diverse approaches distinct from antibiotics or fecal transplant, such as dietary interventions, for mitigating CDI in at-risk human populations. IMPORTANCE Clostridioides difficile is a leading cause of infectious diarrhea in humans, and it imposes a tremendous burden on the health care system. Current treatments for C. difficile infection (CDI) include antibiotics and fecal microbiota transplant, which contribute to recurrent CDIs and face major regulatory hurdles, respectively. Therefore, there is an ongoing need to develop new ways to cope with CDI. Notably, a disrupted "dysbiotic" gut microbiota is the primary risk factor for CDI, but we incompletely understand how a healthy microbiota resists CDI. Here, we show that a specific molecule produced by the gut microbiota, butyrate, is negatively associated with C. difficile burdens in humans and in a mouse model of CDI and that butyrate impedes the growth of diverse C. difficile strains in pure culture. These findings help to build a foundation for designing alternative, possibly diet-based, strategies for mitigating CDI in humans.
Asunto(s)
Clostridioides difficile , Infecciones por Clostridium , Humanos , Animales , Ratones , Butiratos , Tolerancia , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Ácidos Grasos VolátilesRESUMEN
Clostridioides difficile infection (CDI) is routinely diagnosed by PCR, with or without toxin enzyme immunoassay testing. The role of therapy for positive PCR and negative toxin remains unclear. The objective of this study was to determine whether clinical outcomes of PCR+/cycle threshold-based toxin (CT-toxin)- individuals vary by result reporting and treatment strategy. We performed a quasiexperimental noninferiority study comparing clinical outcomes of PCR+/CT-toxin- individuals by reporting PCR result only (most patients treated) with reporting CT-toxin result only (most patients untreated) in a single-center, tertiary academic hospital. The primary outcome was symptomatic PCR+/CT-toxin+ conversion at 8 weeks. Secondary outcomes included 7-day diarrhea resolution, hospital length of stay, and 30-day all-cause mortality. A total of 663 PCR+/CT-toxin- test results were analyzed from 632 individuals with a median age of 61 years (interquartile range [IQR], 44 to 72) and 50.4% immunocompromised. Individuals in the preintervention group were more likely to have received CDI therapy than those in the intervention group (91.5 versus 15.1%; P < 0.001). Symptomatic toxin conversion at 8 weeks and hospital length of stay failed to establish the predefined thresholds for noninferiority. Lack of diarrhea resolution at 7 days and 30-day all-cause mortality was similar and established noninferiority (20.0 versus 13.7%; adjusted odds ratio [aOR], 0.57; 90% confidence interval [CI], 0.32 to 1.01; P = 0.1; and 8.6 versus 6.5%; aOR, 0.46; 90% CI, 0.20 to 1.04; P = 0.12). These data support the safety of withholding antibiotics for selected hospitalized individuals with suspected CDI but negative toxin.
Asunto(s)
Toxinas Bacterianas , Clostridioides difficile , Infecciones por Clostridium , Adulto , Toxinas Bacterianas/análisis , Toxinas Bacterianas/genética , Clostridioides difficile/genética , Infecciones por Clostridium/diagnóstico , Infecciones por Clostridium/tratamiento farmacológico , Diarrea/diagnóstico , Diarrea/tratamiento farmacológico , Heces/química , Humanos , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa/métodosRESUMEN
We report persistent severe acute respiratory syndrome coronavirus 2 infection in a patient with HIV/AIDS; the virus developed spike N terminal domain and receptor binding domain neutralization resistance mutations. Our findings suggest that immunocompromised patients can harbor emerging variants of severe acute respiratory syndrome coronavirus 2.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida , COVID-19 , Humanos , Mutación , Unión Proteica , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus/genéticaRESUMEN
Nucleic acid amplification tests are commonly used to diagnose Clostridioides difficile infection (CDI). Two-step testing with a toxin enzyme immunoassay is recommended to discriminate between infection and colonization but requires additional resources. Prior studies showed that PCR cycle threshold (CT ) can predict toxin positivity with high negative predictive value. Starting in October 2016, the predicted toxin result (CT-toxin) based on a validated cutoff was routinely reported at our facility. To evaluate the clinical efficacy of this reporting, all adult patients with positive GeneXpert PCR results from October 2016 through October 2017 underwent a chart review to measure the recurrence of or conversion to a CT-toxin+ result and 30-day all-cause mortality. There were 482 positive PCR tests in 430 unique patients, 282 CT-toxin+ and 200 CT-toxin- Patient characteristics were similar at testing, though CT-toxin+ patients had higher white blood cell (WBC) counts (12.5 × 103 versus 9.3 × 103 cells/µl; P = 0.001). All cases (n = 21) of fulminant CDI had a CT-toxin+ result. Index CT-toxin+ patients were significantly more likely to have a CT-toxin+ result within 90 days than CT-toxin- patients (17.4% [n = 49] versus 8.0% [n = 16], respectively; P = 0.003). Thirty-day all-cause mortality was higher in CT-toxin- patients (11.1% versus 6.8%; P = 0.1), though no deaths in CT-toxin- patients were directly attributable to CDI. Of the 200 CT-toxin- patients, 51.5% (n = 103) were treated for CDI. The rates of conversion to a CT-toxin+ result (8.8% versus 7.2%; P = 0.8) and all-cause mortality (8.8% versus 13.4%; P = 0.3) were similar between treated and untreated CT-toxin- patients, respectively. CT -based toxin prediction may identify patients at higher risk for CDI-related complications and reduce treatment among CT-toxin- patients.
Asunto(s)
Infecciones por Clostridium/diagnóstico , Infecciones por Clostridium/mortalidad , Enterotoxinas/análisis , Reacción en Cadena de la Polimerasa/estadística & datos numéricos , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Proteínas Bacterianas/genética , Clostridioides difficile/efectos de los fármacos , Clostridioides difficile/genética , Infecciones por Clostridium/tratamiento farmacológico , Infecciones por Clostridium/microbiología , Enterotoxinas/inmunología , Heces/química , Femenino , Humanos , Recuento de Leucocitos , Masculino , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Estudios Retrospectivos , Resultado del Tratamiento , Adulto JovenAsunto(s)
Infecciones por Coronavirus , Máscaras , Pandemias , Neumonía Viral , Betacoronavirus , COVID-19 , Personal de Salud , Humanos , SARS-CoV-2RESUMEN
We used a strand-specific RT-qPCR to evaluate viral replication as a surrogate for infectiousness among 242 asymptomatic inpatients with a positive severe acute respiratory coronavirus virus 2 (SARS-CoV-2) admission test. Only 21 patients (9%) had detectable SARS-CoV-2 minus-strand RNA. Because most patients were found to be noninfectious, our findings support the suspension of asymptomatic admission testing.
Asunto(s)
COVID-19 , Humanos , COVID-19/diagnóstico , COVID-19/epidemiología , SARS-CoV-2/genética , Prueba de COVID-19 , Centros de Atención Terciaria , Técnicas de Laboratorio Clínico , ARN Viral/genéticaRESUMEN
OBJECTIVE: To assess the impact of initial specimen diversion device (ISDD) on inpatient and emergency department blood culture contamination (BCC), central-line-associated bloodstream infection (CLABSI) standardized infection ratios (SIRs), and antibiotic administration. DESIGN: Single-center quasi-experimental prospective cohort study wherein phlebotomists used traditional venipuncture with or without the ISDD while registered nurses (RNs) used traditional venipuncture. METHOD: BCC events among phlebotomists and RNs were observed and compared from March 17, 2019, through January 21, 2020, defined by contaminant detection in 1 of 4 bottles for matched sets or 1 of 2 bottles in both subsets for coagulase negative staphylococci. CLABSIs throughout this period were recorded and SIRs were calculated. Enhanced oversight took place through July 21, 2019, with chart review assessing antibiotic use for patients with possible BCC. RESULTS: Overall, 24% of blood cultures obtained were from patients in intensive care. Phlebotomists using traditional venipuncture (n = 4,759) had a 2.3% BCC rate; phlebotomists using the ISDD (n = 11,202) had a 0% BCC rate. RNs drew 7,411 BCs with a 0.8% BCC rate. The CLABSI SIR was decreased from 1.103 in 2017 and 0.658 in 2018 to 0.439 in 2019. The CLABSI incidence was 33%-64% of predicted value for each 2019 quarter. This range fell to 18%-37% after the exclusion of likely false-positive results. Among 42 patients with possible BCC under enhanced oversight, 2 patients were treated with prolonged antibiotic courses. CONCLUSIONS: ISDD use by phlebotomists was associated with BCC reduction and reduced false-positive CLABSI results. This patient-care quality improvement could constitute sustainable antibiotic stewardship expansion.
Asunto(s)
Infecciones Relacionadas con Catéteres , Cateterismo Venoso Central , Sepsis , Humanos , Cultivo de Sangre/métodos , Estudios Prospectivos , Flebotomía/efectos adversos , Flebotomía/métodos , Antibacterianos/uso terapéutico , Sepsis/etiología , Infecciones Relacionadas con Catéteres/epidemiología , Cateterismo Venoso Central/efectos adversosRESUMEN
Severe acute respiratory coronavirus virus 2 (SARS-CoV-2) real-time reverse-transcription polymerase chain reaction (rRT-PCR) strand-specific assay can be used to identify active SARS-CoV-2 viral replication. We describe the characteristics of 337 hospitalized patients with at least 1 minus-strand SARS-CoV-2 assay performed >20 days after illness onset. This test is a novel tool to identify high-risk hospitalized patients with prolonged SARS-CoV-2 replication.
Asunto(s)
COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , COVID-19/diagnóstico , Replicación Viral , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Helicobacter pylori (Hp) intimately interacts with the gastric epithelial surface and translocates the virulence factor CagA into host cells in a contact-dependent manner. To study how Hp benefits from interacting with the cell surface, we developed live-cell microscopy methods to follow the fate of individual bacteria on the cell surface and find that Hp is able to replicate and form microcolonies directly over the intercellular junctions. On polarized epithelia, Hp is able to grow directly on the apical cell surface in conditions that do not support the growth of free-swimming bacteria. In contrast, mutants in CagA delivery are defective in colonization of the apical cell surface. Hp perturbs the polarized epithelium in a highly localized manner, since wild-type Hp does not rescue the growth defect of the CagA-deficient mutants upon co-infection. CagA's ability to disrupt host cell polarity is a key factor in enabling colonization of the apical cell surface by Hp, as disruption of the atypical protein kinase C/Par1b polarity pathway leads to rescue of the mutant growth defect during apical infection, and CagA-deficient mutants are able to colonize the polarized epithelium when given access to the basolateral cell surface. Our study establishes the cell surface as a replicative niche and the importance of CagA and its effects on host cell polarity for this purpose.
Asunto(s)
Antígenos Bacterianos/metabolismo , Adhesión Bacteriana , Proteínas Bacterianas/metabolismo , Membrana Celular/microbiología , Polaridad Celular , Helicobacter pylori/crecimiento & desarrollo , Animales , Antígenos Bacterianos/genética , Proteínas Bacterianas/genética , Línea Celular , Membrana Celular/metabolismo , Perros , Helicobacter pylori/fisiología , Uniones Intercelulares/metabolismo , Uniones Intercelulares/microbiología , Microscopía Fluorescente , MutaciónRESUMEN
A pan-viral DNA microarray, the Virochip (University of California, San Francisco), was used to detect human parainfluenzavirus 4 (HPIV-4) infection in an immunocompetent adult presenting with a life-threatening acute respiratory illness. The virus was identified in an endotracheal aspirate specimen, and the microarray results were confirmed by specific polymerase chain reaction and serological analysis for HPIV-4. Conventional clinical laboratory testing using an extensive panel of microbiological tests failed to yield a diagnosis. This case suggests that the potential severity of disease caused by HPIV-4 in adults may be greater than previously appreciated and illustrates the clinical utility of a microarray for broad-based viral pathogen screening.
Asunto(s)
Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Virus de la Parainfluenza 4 Humana/aislamiento & purificación , Neumonía Viral/diagnóstico , Infecciones por Rubulavirus/diagnóstico , Adulto , Bronquiolitis Viral/diagnóstico , Femenino , Humanos , Virus de la Parainfluenza 4 Humana/genética , Infecciones por Rubulavirus/virología , Pruebas Serológicas/métodos , Tomógrafos Computarizados por Rayos XRESUMEN
Six cases of coagulase-negative staphylococcal mediastinitis were identified in the latter half of 1999. A new preoperative cleansing solution was suspected by hospital staff to be a factor in the outbreak. We evaluated this possible risk factor along with other known and suspected surgical site infection risk factors in this case-control study.
Asunto(s)
Coagulasa/aislamiento & purificación , Brotes de Enfermedades , Mediastinitis/microbiología , Anciano , Estudios de Casos y Controles , Coagulasa/efectos adversos , Femenino , Cardiopatías/cirugía , Humanos , Masculino , Mediastinitis/etiología , Persona de Mediana Edad , Complicaciones Posoperatorias , Factores de Riesgo , Piel/microbiologíaRESUMEN
An extensive blood culture protocol, including prolonged incubation of cultures, for 215 patients believed to have had endocarditis yielded only 3 clinically relevant results. Twenty-four Haemophilus, Actinobacillus, Cardiobacterium, Eikenella, and Kingella (i.e., HACEK) organisms were recovered from standard 5-day blood cultures during the same time period. Specialized methods and not extended incubation times are recommended for recovery of fastidious agents of septicemia.
Asunto(s)
Bacterias/aislamiento & purificación , Técnicas Bacteriológicas/métodos , Bacteriemia/diagnóstico , Bacteriemia/microbiología , Medios de Cultivo , Endocarditis Bacteriana/diagnóstico , Endocarditis Bacteriana/microbiología , Humanos , Estudios Retrospectivos , Factores de TiempoRESUMEN
We asked whether Campylobacter jejuni isolated from patients with Guillain-Barré syndrome (GBS) differ from isolates isolated from patients with uncomplicated gastrointestinal infection using DNA microarray analysis. We found that specific GBS genes or regions were not identified, and microarray analysis confirmed significant genomic heterogeneity among the isolates.
Asunto(s)
Campylobacter jejuni/genética , Enteritis/microbiología , Genoma Bacteriano , Síndrome de Guillain-Barré/microbiología , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Variación Genética , HumanosRESUMEN
To extend our knowledge of host-cell targets of Helicobacter pylori, we characterized the interaction between H. pylori and human T84 epithelial cell polarized monolayers. Transcriptional analysis by use of human microarrays and a panel of isogenic H. pylori mutants revealed distinct responses to infection. Of the 670 genes whose expression changed, most (92%) required the cag pathogenicity island (PAI). Although altered expression of many genes was dependent on CagA (80% of the PAI-dependent genes), expression of >30% of these host genes occurred independent of the phosphorylation state of the CagA protein. Similarly, we found that injected CagA localized to the apical surface of cells and showed preferential accumulation at the apical junctions in a phosphorylation-independent manner. These data suggest the presence of distinct functional domains within the CagA protein that play essential roles in protein targeting and alteration of host-cell signaling pathways.
Asunto(s)
Antígenos Bacterianos/metabolismo , Proteínas Bacterianas/metabolismo , Línea Celular Tumoral/microbiología , Helicobacter pylori/metabolismo , Factores de Virulencia/metabolismo , Antígenos Bacterianos/genética , Proteínas Bacterianas/genética , Línea Celular Tumoral/fisiología , Polaridad Celular , Forma de la Célula , Regulación de la Expresión Génica , Helicobacter pylori/genética , Helicobacter pylori/patogenicidad , Humanos , Microscopía Confocal , Mutación , Análisis de Secuencia por Matrices de Oligonucleótidos , Fosforilación , Transducción de Señal , Factores de Virulencia/genéticaRESUMEN
Helicobacter pylori infects over half the world's population and causes a wide range of diseases, including gastritis, peptic ulcer, and two forms of gastric cancer. H. pylori infection elicits a variety of phenotypic responses in cultured gastric epithelial cells, including the expression of proinflammatory genes and changes in the actin cytoskeleton. Both of these responses are mediated by the type IV secretion system (TFSS) encoded by the cag pathogenicity island (cag PAI). We used human cDNA microarrays to examine the temporal transcriptional profiles of gastric AGS cells infected with H. pylori strain G27 and a panel of isogenic mutants to dissect the contributions of various genes in the cag PAI. Infection with G27 induced expression of genes involved in the innate immune response, cell shape regulation, and signal transduction. A mutant lacking the cagA gene, which encodes an effector molecule secreted by the TFSS and required for the host cell cytoskeletal response, induced the expression of fewer cytoskeletal genes. A mutant lacking cagE, which encodes a structural component of the TFSS, failed to up-regulate a superset of host genes, including the cagA-dependent genes, and many of the immune response genes. A mutant lacking the entire cag PAI failed to induce both the cagE-dependent genes and several transiently expressed cagE independent genes. Host cell transcriptional profiling of infection with isogenic strains offered a detailed molecular picture of H. pylori infection and provided insight into potential targets of individual virulence determinants such as tyrosine kinase and Rho GTPase signaling molecules.
Asunto(s)
Proteínas Bacterianas/genética , Mucosa Gástrica/microbiología , Helicobacter pylori/genética , Helicobacter pylori/patogenicidad , Virulencia/genética , Regulación Viral de la Expresión Génica , Genotipo , Infecciones por Helicobacter/microbiología , Humanos , Mutagénesis , Análisis de Secuencia por Matrices de Oligonucleótidos , Transcripción GenéticaRESUMEN
Although intracellular Helicobacter pylori have been described in biopsy specimens and in cultured epithelial cells, the fate of these bacteria is unknown. Using differential interference contrast (DIC) video and immunofluorescence microscopy, we document that a proportion of cell-associated H. pylori enter large cytoplasmic vacuoles, where they remain viable and motile and can survive lethal concentrations of extracellular gentamicin. Entry into vacuoles occurs in multiple epithelial cell lines including AGS gastric adenocarcinoma, Caco-2 colon adenocarcinoma and MDCK kidney cell line, and depends on the actin cytoskeleton. Time-lapse microscopy over several hours was used to follow the movement of live H. pylori within vacuoles of a single cell. Pulsed, extracellular gentamicin treatments show that the half-life of intravacuolar bacteria is on the order of 24 h. Viable H. pylori repopulate the extracellular environment in parallel with the disappearance of intravacuolar bacteria, suggesting release from the intravacuolar niche. Using electron microscopy and live fluorescent staining with endosomal dyes, we observe that H. pylori-containing vacuoles are similar in morphology to late endosomal multivesicular bodies. VacA is not required for these events, as isogenic vacA- mutants still enter and survive within the intravacuolar niche. The exploitation of an intravacuolar niche is a new aspect of the biological life cycle of H. pylori that could explain the difficulties in eradicating this infection.
Asunto(s)
Células Epiteliales/microbiología , Infecciones por Helicobacter/microbiología , Helicobacter pylori/fisiología , Vacuolas/microbiología , Animales , Antibacterianos/farmacología , Antígenos Bacterianos/metabolismo , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/metabolismo , Línea Celular , Citocalasina D/metabolismo , Citoesqueleto/metabolismo , Endocitosis , Células Epiteliales/citología , Células Epiteliales/metabolismo , Colorantes Fluorescentes/metabolismo , Gentamicinas/farmacología , Infecciones por Helicobacter/patología , Helicobacter pylori/efectos de los fármacos , Helicobacter pylori/ultraestructura , Humanos , Concentración de Iones de Hidrógeno , Microscopía Confocal , Microscopía por Video , Células Tumorales Cultivadas , Vacuolas/metabolismo , Vacuolas/ultraestructuraRESUMEN
Colonization by the gastric pathogen Helicobacter pylori has been shown to be intricately linked to the development of gastritis, ulcers, and gastric malignancy. Little is known about mechanisms employed by the bacterium that help it adapt to the hostile environment of the human stomach. In an effort to extend our knowledge of these mechanisms, we utilized spotted-DNA microarrays to characterize the response of H. pylori to low pH. Expression of approximately 7% of the bacterial genome was reproducibly altered by shift to low pH. Analysis of the differentially expressed genes led to the discovery that acid exposure leads to profound changes in motility of H. pylori, as a larger percentage of acid-exposed bacterial cells displayed motility and moved at significantly higher speeds. In contrast to previous publications, we found that expression of the bacterial virulence gene cagA was strongly repressed by acid exposure. Furthermore, this transcriptional repression was reflected at the level of protein accumulation in the H. pylori cell.
Asunto(s)
Regulación Bacteriana de la Expresión Génica , Helicobacter pylori/genética , Amoníaco/metabolismo , Animales , Antígenos Bacterianos/genética , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas Bacterianas/genética , Flagelina/biosíntesis , Gerbillinae , Helicobacter pylori/fisiología , Humanos , Concentración de Iones de Hidrógeno , Masculino , Análisis de Secuencia por Matrices de Oligonucleótidos , Regulación hacia ArribaRESUMEN
Iron is an essential nutrient that is often found in extremely limited available quantities within eukaryotic hosts. Because of this, many pathogenic bacteria have developed regulated networks of genes important for iron uptake and storage. In addition, it has been shown that many bacteria use available iron concentrations as a signal to regulate virulence gene expression. We have utilized DNA microarray technology to identify genes of the human pathogen Helicobacter pylori that are differentially regulated on a growth-inhibiting shift to iron starvation conditions. In addition, the growth phase-dependent expression of these genes was investigated by examining both exponential and stationary growth phase cultures. We identified known iron-regulated genes, as well as a number of genes whose regulation by iron concentration was not previously appreciated. Included in the list of regulated factors were the known virulence genes cagA, vacA, and napA. We examined the effect of iron starvation on the motility of H. pylori and found that exponential- and stationary-phase cultures responded differently to the stress. We further found that while growing cells are rapidly killed by iron starvation, stationary-phase cells show a remarkable ability to survive iron depletion. Finally, bioinformatic analysis of the predicted promoter regions of the differentially regulated genes led to identification of several putative Fur boxes, suggesting a direct role for Fur in iron-dependent regulation of these genes.