RESUMEN
Better diagnostic tools are needed to improve the diagnosis of Clostridioides difficile infections (CDI) and reduce the overtreatment of colonized children. In this study, we evaluated two polymerase chain reaction (PCR) assays (Cepheid GeneXpert C. difficile and the Gastroenteritis PCR Panel by QIAstat-Dx) as a standalone method in combination with the PCR cycle threshold (Ct) value in positive samples to predict the presence of free toxins. We also evaluated the clinical impact of reporting toxin production results and provided comments alongside the PCR results in our pediatric population. PCR-positive stool samples from pediatric patients (aged 2 to 18 years old) were included in our study and tested for the presence of toxins A and B using the C. difficile Quik Chek Complete kit. For the clinical intervention, the CDI treatment rates 6 months pre- and post-intervention were compared. The use of PCR Ct value showed excellent sensitivity (100%) at a Ct value cutoff of 26.1 and 27.2 using the Cepheid GeneXpert C. difficile and the Gastroenteritis PCR Panel by QIAstat-Dx, respectively, while the toxin test showed inferior sensitivity of 64% in the PCR-positive samples. In addition, CDI treatment rates were decreased by 23% post-intervention. The results of our study suggest that nucleic acid amplification test (NAAT) assays supplemented by the use of PCR Ct value for positive samples can be used as standalone tests to differentiate CDI from colonization. Furthermore, the reporting of toxin production along with the PCR results can help reduce the unnecessary treatment of colonized children.
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The Indian River Lagoon (IRL), a 156-mile-long estuary located on the eastern coast of Florida, experiences phytoplankton bloom events due to increased seasonal temperatures coupled with anthropogenic impacts. This study aimed to gather data on the toxicity to human cells and to identify secondary metabolites found in water samples collected in the IRL. Water samples from 20 sites of the IRL were collected during the wet and dry seasons over a three-year period. A panel of cell lines was used to test cytotoxicity. Hemagglutination, hemolysis, and inhibition of protein phosphatase 2A (PP2A) were also measured. Cytotoxic blooms were seen both in the south (Microcystis) and the north (Pyrodinium) of the IRL. Each toxin induced a consistent pattern of cytotoxicity in the panel of human cell lines assayed. During blooms, cytotoxicity due to a single type of toxin is obvious from this pattern. In the absence of blooms, the cytotoxicity seen reflected either a mixture of toxins or it was caused by an unidentified toxin. These observations suggest that other toxins with the potential to be harmful to human health may be present in the IRL. Moreover, the presence of toxins in the IRL is not always associated with blooms of known toxin-producing organisms.
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Dinoflagelados , Toxinas Biológicas , Humanos , Ríos , Dinoflagelados/fisiología , Eutrofización , Saxitoxina , Agua , Floraciones de Algas NocivasRESUMEN
Cryptosporidium sp. are apicomplexan parasites that cause significant morbidity and possible mortality in humans and valuable livestock. There are no drugs on the market that are effective in the population most severely affected by this parasite. This study is the first high-throughput screen for potent anti-Cryptosporidium natural products sourced from a unique marine compound library. The Harbor Branch Oceanographic Institute at Florida Atlantic University has a collection of diverse marine organisms some of which have been subjected to medium pressure liquid chromatography to create an enriched fraction library. Numerous active compounds have been discovered from this library, but it has not been tested against Cryptosporidium parvum. A high-throughput in vitro growth inhibition assay was used to test 3764 fractions in the library, leading to the identification of 23 fractions that potently inhibited the growth of Cryptosporidium parvum. Bioassay guided fractionation of active fractions from a deep-sea sponge, Leiodermatium sp., resulted in the purification of leiodolide A, the major active compound in the organism. Leiodolide A displayed specific anti-Cryptosporidium activity at a half maximal effective concentration of 103.5 nM with selectivity indexes (SI) of 45.1, 11.9, 19.6 and 14.3 for human ileocecal colorectal adenocarcinoma cells (HCT-8), human hepatocellular carcinoma cells (Hep G2), human neuroblastoma cells (SH-SY5Y) and green monkey kidney cells (Vero), respectively. The unique structure of leiodolide A provides a valuable drug scaffold on which to develop new anti-Cryptosporidium compounds and supports the importance of screening natural product libraries for new chemical scaffolds.
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Productos Biológicos , Criptosporidiosis , Cryptosporidium parvum , Cryptosporidium , Animales , Productos Biológicos/farmacología , Línea Celular , Chlorocebus aethiops , Criptosporidiosis/parasitología , Ensayos Analíticos de Alto Rendimiento , HumanosRESUMEN
Clam Beach is located in Northern California, USA, and is listed as an impaired waterway by the federal government. The scope of this study was to investigate this beach and surrounding watershed to determine, if possible, the source of the impairment by conducting an 11-h beach study and 8-week watershed study. We used traditional fecal indicator bacteria (FIB) and microbial source tracking (MST) methods to help identify source(s) of the FIB. Our study was focused on four possible contributors: human, ruminant, canine, and bird. A total of 169 samples were collected, analyzed, and compared to the California Department of Health single sample maximum (SSM) objective. In the beach study, 29 (44%) samples exceeded at least one SSM objective, which would have resulted in a resample per state regulations for recreational primary contact use. MST methods showed that the most abundant marker detected was bird, in 65% of the samples, but varied by sample location, which is likely due to a natural population of nearshore birds regularly observed along Clam Beach. The watershed study highlighted the potential influence from ruminants throughout the region, while humans did not appear to be a significant contributor. Health risk to humans appears to be low.
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Bivalvos , Animales , Playas , California , Perros , Monitoreo del Ambiente , Heces , Humanos , Microbiología del AguaRESUMEN
Novel drug leads for malaria therapy are urgently needed because of the widespread emergence of resistance to all available drugs. Screening of the Harbor Branch enriched fraction library against the Plasmodium falciparum chloroquine-resistant strain (Dd2) followed by bioassay-guided fractionation led to the identification of two potent antiplasmodials; a novel diterpene designated as bebrycin A (1) and the known C21 degraded terpene nitenin (2). A SYBR Green I assay was used to establish a Dd2 EC50 of 1.08 ± 0.21 and 0.29 ± 0.02 µM for bebrycin A and nitenin, respectively. Further analysis was then performed to assess the stage specificity of the inhibitors antiplasmodial effects on the Dd2 intraerythrocytic life cycle. Exposure to bebrycin A was found to block parasite maturation at the schizont stage if added any time prior to late schizogony at 42 hours post invasion, (HPI). In contrast, early life cycle exposure to nitenin (prior to 18 HPI) was identified as crucial to parasite inhibition, suggesting nitenin may target the maturation of the parasite during the transition from ring to early trophozoite (6-18 HPI), a novel property among known antimalarials.
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Antozoos/metabolismo , Antimaláricos/farmacología , Diterpenos/farmacología , Malaria Falciparum/tratamiento farmacológico , Plasmodium falciparum/efectos de los fármacos , Poríferos/metabolismo , Animales , Antimaláricos/aislamiento & purificación , Diterpenos/aislamiento & purificación , Células Hep G2 , Humanos , Estadios del Ciclo de Vida , Malaria Falciparum/parasitología , Estructura Molecular , Plasmodium falciparum/crecimiento & desarrollo , Relación Estructura-Actividad , Factores de TiempoRESUMEN
Tuberculosis is the leading cause of death due to infectious disease worldwide. There is an urgent need for more effective compounds against this pathogen to control the disease. Investigation of the anti-mycobacterial activity of a deep-water sponge of the genus Plakina revealed the presence of a new steroidal alkaloid of the plakinamine class, which we have given the common name plakinamine P. Its structure is most similar to plakinamine L, which also has an acyclic side chain. Careful dissection of the nuclear magnetic resonance data, collected in multiple solvents, suggests that the dimethyl amino group at the 3 position is in an equatorial rather than axial position unlike previously reported plakinamines. Plakinamine P was bactericidal against M. tuberculosis, and exhibited moderate activity against other mycobacterial pathogens, such as M. abscessus and M. avium. Furthermore, it had low toxicity against J774 macrophages, yielding a selectivity index (SI, or IC50/MIC) of 8.4. In conclusion, this work provides a promising scaffold to the tuberculosis drug discovery pipeline. Future work to determine the molecular target of this compound may reveal a pathway essential for M. tuberculosis survival during infection.
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Alcaloides/química , Alcaloides/farmacología , Antituberculosos/farmacología , Mycobacterium tuberculosis/efectos de los fármacos , Esteroides/química , Esteroides/farmacología , Antituberculosos/química , Estructura MolecularRESUMEN
Two new analogues of the potent antitumor compound leiodermatolide, which we call leiodermatolides B and C, have been isolated from specimens of a deep-water sponge of the genus Leiodermatium collected off Florida. The compounds were purified using standard chromatographic methods, and the structures defined through interpretation of the HRMS and 1D and 2D NMR data. Leiodermatolide B (2) lacks the C-21 hydroxy group found in leiodermatolide and has equal potency as the parent compound, providing a simpler analogue for possible clinical development. It inhibits the proliferation of the AsPC-1 human pancreatic adenocarcinoma cell line with an IC50 of 43 nM. Leiodermatolide C (3) has a modified macrolide ring and is over 85-fold less potent with an IC50 of 3.7 µM against the same cell line. These compounds add to the knowledge of the pharmacophore of this class of potent antitumor agents.
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Antineoplásicos/aislamiento & purificación , Antineoplásicos/farmacología , Macrólidos/aislamiento & purificación , Macrólidos/farmacología , Poríferos/química , Animales , Antineoplásicos/química , Ensayos de Selección de Medicamentos Antitumorales , Florida , Humanos , Macrólidos/química , Estructura MolecularRESUMEN
During the 2012-2013 school year, only 66% of students at a Northern Indiana High School were in compliance with school immunization requirements. We report here successful implementation of evidence-based, time, and cost-effective methods aimed at increasing school immunization compliance. A three-stage strategy initiated by the school nurse was employed. In the first stage, letters were sent home with students, indicating the lack of compliance with school immunization laws. The next stage involved a second letter sent home with the student which contained immunization information from the Indiana State Department of Health, appointment information, and a copy of the student's immunization record. In the final stage, letters were sent home via e-mail and phone calls were used for follow-up. At each stage, students and parents were given an explanation of exclusion and a date when exclusion would apply. Postintervention, vaccine compliance was 99.6%, exceeding both national and state averages.
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Medicina Basada en la Evidencia/métodos , Programas de Inmunización/métodos , Evaluación de Programas y Proyectos de Salud , Servicios de Salud Escolar , Servicios de Enfermería Escolar/métodos , Vacunas/uso terapéutico , Adolescente , Femenino , Adhesión a Directriz , Humanos , Indiana , MasculinoRESUMEN
Methicillin-resistant Staphylococcus aureus contributes significantly to cost, morbidity, and mortality due to infectious disease. We surveyed community-associated MRSA isolates to determine which strains were present within anatomical sites of interest. The most likely sources of MRSA among anatomic sites swabbed were wounds followed by the nasal cavity. The USA 300 MRSA strain was most commonly isolated among wound infections while nasal swabs largely yielded USA 100 MRSA. The frequency of isolation of USA 100 amongst community-associated strains is clinically significant as this strain is often correlated with invasive disease, exhibits broad antibiotic resistance, and has been considered to be hospital associated. The potential of USA 100 to cause serious disease and the frequency of its isolation suggest an important reservoir for opportunistic infection. These data demonstrate that MRSA epidemic clones are widespread among the community.
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Infecciones Comunitarias Adquiridas/epidemiología , Infecciones Comunitarias Adquiridas/microbiología , Staphylococcus aureus Resistente a Meticilina/clasificación , Técnicas de Tipificación Bacteriana , Células Clonales , Florida/epidemiología , Humanos , Staphylococcus aureus Resistente a Meticilina/aislamiento & purificaciónRESUMEN
We present the draft genome sequence of methicillin-resistant Staphylococcus aureus strain CBD-635, from the USA100 lineage. This is a sepsis isolate obtained from Tampa General Hospital. This strain is spa type t003 and multilocus sequence typing (MLST) type ST5, and it has been used by our group in the study of novel antimicrobial chemotherapeutics.
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BACKGROUND: We characterized 100 USA100 epidemic MRSA from individuals in the community with no known healthcare-associated risk factors. FINDINGS: Molecular epidemiology demonstrated 21 pulsed-field types and six spa types. SCCmec typing demonstrated that all of the strains possess the type II cassette. The staphylococcal enterotoxin D virulence gene was also present. CONCLUSION: Characterization of USA100 MRSA in the community illustrated the importance of nasal carriage, and the genetic diversity of the USA100 clone.
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Antimitóticos/química , Macrólidos/química , Poríferos/química , Animales , Antimitóticos/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Macrólidos/farmacología , Mitosis/efectos de los fármacos , Estructura Molecular , Resonancia Magnética Nuclear Biomolecular , Océanos y MaresRESUMEN
A reported loss of mecA prompted us to monitor 360 cryostocked methicillin-resistant Staphylococcus aureus strains for stability. Concurrently, 14 well-characterized strains were stored in a Microbank preservation system and subjected to multiple freeze-thaw events. There were no significant declines in the methicillin-resistant populations with either method over a two-year period.
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Proteínas Bacterianas/genética , Resistencia a la Meticilina , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/genética , Recuento de Colonia Microbiana , Congelación , Eliminación de Gen , Proteínas de Unión a las PenicilinasRESUMEN
Recent studies have described a number of fatalities due to methicillin-resistant Staphylococcus aureus (MRSA) and influenza virus co-infection. MRSA isolates provide a challenge to caregivers due to inherent wide range antibiotic resistance. Many facilities have instituted screening methods, based on the presence of antibiotic resistance genes, to identify MRSA positive patients upon admission. However, the resistance profile of the pathogen does not necessarily determine the severity of disease caused by that organism. We describe a fatal case of necrotizing pneumonia in a patient co-infected with Influenza B and a community-associated, PVL-positive methicillin-susceptible Staphylococcus aureus (MSSA).
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Resistencia a la Meticilina , Neumonía Estafilocócica/tratamiento farmacológico , Adulto , Autopsia , Toxinas Bacterianas , Sangre/microbiología , Sangre/virología , Bronconeumonía/tratamiento farmacológico , Dermatoglifia del ADN , Electroforesis en Gel de Campo Pulsado , Exotoxinas , Resultado Fatal , Femenino , Humanos , Virus de la Influenza B/genética , Virus de la Influenza B/aislamiento & purificación , Gripe Humana/complicaciones , Gripe Humana/patología , Leucocidinas , Pulmón/microbiología , Pulmón/patología , Necrosis , Neumonía Estafilocócica/complicaciones , Neumonía Estafilocócica/patología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/genética , Staphylococcus aureus/aislamiento & purificaciónRESUMEN
Pulsed-field gel electrophoresis (PFGE) is currently the gold standard for methicillin-resistant Staphylococcus aureus (MRSA) typing but only one enzyme, SmaI, is currently used for restriction digest. We report the use of virtual digestion to identify enzymes for S. aureus PFGE. Two enzymes (EagI and SacII) were identified and successfully used to characterize two sets of S. aureus isolates, 12 USA300, and 14 additional MRSA isolates comprised of seven SmaI patterns. Phylogenetic analysis of patterns generated by all enzymes determined that the USA300 MRSAs are identical. In contrast, digestion with EagI or SacII resolved one to two band differences among three MRSA pattern sets that were not detected using SmaI. These results demonstrate that a second enzyme may detect differences in S. aureus isolates not detected by single enzyme digestion. However, because isolates differing by one to two bands are considered identical, such discrimination may not be clinically or epidemiologically relevant.
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Electroforesis en Gel de Campo Pulsado/métodos , Resistencia a la Meticilina , Staphylococcus aureus/clasificación , Simulación por Computador , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Humanos , Mapeo Restrictivo/métodosRESUMEN
We examined 299 methicillin-resistant, community-associated Staphylococcus aureus isolates from Florida and Washington State for the presence of the USA300 epidemic clone. Pulsed-field gel electrophoresis demonstrated the epidemic clone in 43% of our S. aureus strains and in isolates from both states. The majority of the USA300 isolates (88%) were from wound infections.
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Infecciones Comunitarias Adquiridas/microbiología , Resistencia a la Meticilina , Meticilina/farmacología , Infecciones Estafilocócicas/epidemiología , Staphylococcus aureus/efectos de los fármacos , Infecciones Comunitarias Adquiridas/epidemiología , Farmacorresistencia Bacteriana , Electroforesis en Gel de Campo Pulsado , Florida/epidemiología , Humanos , Staphylococcus aureus/aislamiento & purificación , Washingtón/epidemiología , Infección de Heridas/epidemiología , Infección de Heridas/microbiologíaRESUMEN
We examined 215 Enterococcus faecalis isolates and found that neither the two-component regulatory locus fsr (E. faecalis regulator) nor gelatinase production was more common in disease-associated isolates than in isolates colonizing healthy individuals (ca. 60 to 65%). The majority of gelatinase-negative isolates, including 14 endocarditis isolates (of 80 isolates tested), contained the previously described 23.9-kb deletion and lacked fsrA and fsrB. While these findings indicate that neither fsr nor gelatinase is required for E. faecalis to cause infection, this study did not address whether fsr or gelatinase affects the severity of disease, as it does in animal models.