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INTRODUCTION: Bacterial sexually transmitted infections (STIs) pose a major public health problem. The emergence of antibiotic-resistant strains of Neisseria gonorrhoeae represents a serious threat to successful treatment and epidemiological control. The first extensively drug-resistant (XDR) strains (ceftriaxone-resistant and high-level azithromycin-resistant [HLR AZY]) have been reported. AIMS: To identify molecular mechanisms implicated in azithromycin resistance in strains isolated from patients over a three-year period in a university hospital in Switzerland. MATERIAL AND METHODS: From January 2020 to December 2022, 34 isolates (one per patient) were recovered from samples analyzed at the University Hospital of Lausanne. Eight genes involved in azithromycin resistance were sequenced: mtrR repressor (mtrCDE operon repressor) and his promotor mtrR-pr, rplD gene (L4 ribosomal protein), rplV gene (L22 ribosomal protein) and the four alleles of the rrl gene (23S rRNA). RESULTS: With a cutoff value of 1 mg/L, 15 isolates were considered as being resistant to azithromycin, whereas the remaining 19 were susceptible. The C2597T mutation in 3 or 4 of the rrl allele confer a medium-level resistance to azithromycin (MIC = 16 mg/L, N = 2). The following mutations were significantly associated with MIC values ≥1 mg/L: the three mutations V125A, A147G, R157Q in the rplD gene (N = 10) and a substitution A->C in the mtrR promotor (N = 9). Specific mutations in the mtrR repressor and its promotor were observed in both susceptible and resistant isolates. CONCLUSIONS: Resistance to azithromycin was explained by the presence of mutations in many different copies of 23S RNA ribosomal genes and their regulatory genes. Other mutations, previously reported to be associated with azithromycin resistance, were documented in both susceptible and resistant isolates, suggesting they play little role, if any, in azithromycin resistance.
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Antibacterianos , Azitromicina , Proteínas Bacterianas , Farmacorresistencia Bacteriana , Mutación , Neisseria gonorrhoeae , Proteínas Represoras , Azitromicina/farmacología , Neisseria gonorrhoeae/genética , Neisseria gonorrhoeae/efectos de los fármacos , Humanos , Proteínas Represoras/genética , Farmacorresistencia Bacteriana/genética , Proteínas Bacterianas/genética , Antibacterianos/farmacología , Pruebas de Sensibilidad Microbiana , Proteínas Ribosómicas/genética , Gonorrea/microbiología , Gonorrea/tratamiento farmacológico , Masculino , FemeninoRESUMEN
PURPOSE: Traditional epidemiological investigations of healthcare-associated Clostridioides difficile infection (HA-CDI) are often insufficient. This study aimed to evaluate a procedure that includes secondary isolation and genomic typing of single toxigenic colonies using core genome multilocus sequence typing (cgMLST) for the investigation of C. difficile transmission. METHODS: We analyzed retrospectively all toxigenic C. difficile-positive stool samples stored at the Lausanne University Hospital over 6 consecutive months. All isolates were initially typed and classified using a modified double-locus sequence typing (DLST) method. Genome comparison of isolates with the same DLST and clustering were subsequently performed using cgMLST. The electronic administrative records of patients with CDI were investigated for spatiotemporal epidemiological links supporting hospital transmission. A comparative descriptive analysis between genomic and epidemiological data was then performed. RESULTS: From January to June 2021, 86 C. difficile isolates were recovered from thawed samples of 71 patients. Thirteen different DLST types were shared by > 1 patient, and 13 were observed in single patients. A genomic cluster was defined as a set of isolates from different patients with ≤ 3 locus differences, determined by cgMLST. Seven genomic clusters were identified, among which plausible epidemiological links were identified in only 4/7 clusters. CONCLUSION: Among clusters determined by cgMLST analysis, roughly 40% included unexplained HA-CDI acquisitions, which may be explained by unidentified epidemiological links, asymptomatic colonization, and/or shared common community reservoirs. The use of DLST, followed by whole genome sequencing analysis, is a promising and cost-effective stepwise approach for the investigation of CDI transmission in the hospital setting.
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Clostridioides difficile , Infecciones por Clostridium , Humanos , Tipificación de Secuencias Multilocus/métodos , Clostridioides difficile/genética , Clostridioides/genética , Estudios Retrospectivos , Infecciones por Clostridium/epidemiología , Infecciones por Clostridium/microbiología , Hospitales , Genoma BacterianoRESUMEN
PURPOSE: Toxigenic Clostridioides difficile is responsible for up to one third of post antibiotic diarrhea and for more than 95% of pseudomembranous colitis. Nowadays, diagnosis relies on the documentation of the presence of the toxin in stools by specific antigenic or PCR tests. Stool cultures have been mostly abandoned, leading to the absence of isolates for further epidemiological analyses. METHODS: Aliquots of stool samples, frozen for up to two years, were thawed and inoculated onto commercial C. difficile media. Eighteen stools were recovered from patients hospitalized in the pediatric ward where at that time a chain of transmission was suspected. Eleven stools were recovered from patients hospitalized in a medical ward over a three months period with no suspected transmission event. Up to 16 characteristic colonies were isolates per culture. PCR of toxins genes and molecular typing by Double Locus Sequence Typing (DLST) were performed on these colonies. Whole genome multi locus sequence typing (wgMLST) was performed on selected isolates. RESULTS: Among the 29 stool specimens, no growth was observed for four stools and only one colony grew for one stool. Except the latter, all 16 colonies of the 24 stools showed identical toxin genes profiles than the original stool. However, variant DLST genotypes was observed within 20% of investigated stools. The majority of variants were single locus variant due to an IN/DEL of the repeat in one of the two DLST locus. Despite this variation, results of molecular typing overrule the putative transmission chain in the pediatric ward and revealed undetected chains of transmission in the medical ward. These results were confirmed with wgMLST. CONCLUSIONS: The developed protocol allows prospective and retrospective molecular and genomic epidemiological investigation of C. difficile infections for infection control purpose.
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Clostridioides difficile , Infecciones por Clostridium , Antibacterianos , Niño , Clostridioides , Clostridioides difficile/genética , Infecciones por Clostridium/diagnóstico , Heces , Humanos , Tipificación de Secuencias Multilocus/métodos , Estudios Prospectivos , Estudios RetrospectivosRESUMEN
Features of Clostridioides difficile transmission in swine and the role of rodents as C. difficile reservoir are not clear. To investigate if rodents can carry strains of C. difficile that are genetically similar to those isolated from swine, 97 fecal samples from neonatal piglets and 41 intestinal contents from rodents were collected in two farms. All samples were subjected to C. difficile culture and the presence of A/B toxins in piglet feces were accessed by commercial enzyme imunoassay (EIA). C. difficile isolates were typed by double- (DLST) and multi-locus sequence typing (MLST). C. difficile was isolated from 15.5% of piglets and 31.7% of rodents. Most isolates were identified as DLST type 4-4 and 17-5 (both are ST11), which were found in both rodents and piglets. Results of this study suggested that rodents may have a role on the transmission and spread of C. difficile strains to swine.
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Portador Sano/veterinaria , Clostridioides difficile/aislamiento & purificación , Infecciones por Clostridium/veterinaria , Reservorios de Enfermedades/microbiología , Roedores/microbiología , Enfermedades de los Porcinos/microbiología , Animales , Toxinas Bacterianas/análisis , Técnicas Bacteriológicas , Portador Sano/microbiología , Clostridioides difficile/clasificación , Clostridioides difficile/genética , Infecciones por Clostridium/microbiología , Heces/microbiología , Genotipo , Técnicas para Inmunoenzimas , Tipificación de Secuencias Multilocus , PorcinosRESUMEN
The claustrum is a small, elongated nucleus close to the external capsule and deep in the insular cortex. In rodents, this nucleus is characterized by a dense cluster of parvalbumin labeling. The claustrum is connected with the cerebral cortex. It does not project to the brainstem, but brainstem structures can influence this nucleus. To identify some specific projections from the lateral hypothalamus and midbrain, we analyzed the distribution of projections labeled with antibodies against tyrosine hydroxylase (TH), melanin-concentrating hormone (MCH), and hypocretin (Hcrt) in the region of the claustrum. The claustrum contains a significant projection by MCH axons, whereas it is devoid of TH projections. Unlike TH and MCH axons, Hcrt axons are scattered throughout the region. This observation is discussed mainly with regard to the role of the claustrum in cognitive functions and that of MCH in REM sleep. J. Comp. Neurol. 525:1489-1498, 2017. © 2016 Wiley Periodicals, Inc.
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Axones/metabolismo , Ganglios Basales/citología , Ganglios Basales/metabolismo , Vías Nerviosas/citología , Vías Nerviosas/metabolismo , Animales , Técnica del Anticuerpo Fluorescente , Hormonas Hipotalámicas/metabolismo , Imagenología Tridimensional , Masculino , Melaninas/metabolismo , Orexinas/metabolismo , Hormonas Hipofisarias/metabolismo , Ratas , Ratas Sprague-Dawley , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
Neurons producing the melanin-concentrating hormone (MCH) are distributed in the posterior hypothalamus, but project massively throughout the forebrain. Many aspects regarding the anatomical organization of these projections are still obscure. The present study has two goals: first to characterize the topographical organization of neurons projecting into the cholinergic basal forebrain (globus pallidus, medial septal complex), and second to verify if MCH neurons may indirectly influence the dorsal striatum (caudoputamen) by innervating afferent sources to this structure. In the first series of experiments, the retrograde tracer fluorogold was injected into multiple sites in the pallidal and medial septal regions and the distribution of retrogradely labeled neurons were analyzed in the posterior lateral hypothalamus. In the second series of experiments, fluorogold was injected into the caudoputamen, and the innervation by MCH axons of retrogradely labeled cells was analyzed. Our results revealed that the MCH system is able to interact with the basal nuclei in several different ways. First, MCH neurons provide topographic inputs to the globus pallidus, medial septal complex, and substantia innominata. Second, striatal projecting neurons in the cortex, thalamus, and substantia nigra presumably receive only sparse inputs from MCH neurons. Third, the subthalamic nucleus is heavily innervated by MCH projections, thus, presumably serves as one important intermediate station to mediate MCH influence on other parts of the basal nuclei.
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Alcohol consumption during pregnancy can cause a "fetal alcoholic syndrome" (FAS) in the progeny. This syndrome is characterized by important brain defects often associated to a decreased expression of the morphogenic protein sonic hedgehog (Shh). The goal of this study was to verify if a FAS could modify the differentiation of hypothalamic neurons producing MCH. Indeed, the expression of this peptide and neurons producing it are dependent of a Shh controlled genetic cascade in the embryo. To address this question, female rats received a 15% ethanol solution to drink during pregnancy and lactation. Higher abortion rate and smaller pups at birth confirmed that descendants were affected by this experimental condition. MCH expression was analyzed by RT-qPCR and immunohistochemistry in embryos taken at E11 and E13, or in pups and young adults born from control and alcoholic mothers. MCH expression level, number of MCH neurons or ratio of MCH sub-populations were not modified by our experimental conditions. However, Shh expression was significantly lover at E11 and we also observed that hindbrain serotonergic neurons were affected as reported in the literature. These findings as well as other data from the literature suggest that protective mechanisms are involved to maintain peptide expressions and differentiation of some specific neuron populations in the ventral diencephalon in surviving embryos exposed to ethanol during pregnancy.
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Etanol/toxicidad , Trastornos del Espectro Alcohólico Fetal/metabolismo , Proteínas Hedgehog/metabolismo , Hormonas Hipotalámicas/metabolismo , Hipotálamo/efectos de los fármacos , Hipotálamo/metabolismo , Melaninas/metabolismo , Neuronas/metabolismo , Hormonas Hipofisarias/metabolismo , Animales , Peso Corporal/efectos de los fármacos , Femenino , Hipotálamo/embriología , Embarazo , Ratas , Ratas Sprague-Dawley , Serotonina/metabolismoRESUMEN
Sub-populations of neurons producing melanin-concentrating hormone (MCH) are characterized by distinct projection patterns, birthdates and CART/NK3 expression in rat. Evidence for such sub-populations has not been reported in other species. However, given that genetically engineered mouse lines are now commonly used as experimental models, a better characterization of the anatomy and morphofunctionnal organization of MCH system in this species is then necessary. Combining multiple immunohistochemistry experiments with in situ hybridization, tract tracing or BrdU injections, evidence supporting the hypothesis that rat and mouse MCH systems are not identical was obtained: sub-populations of MCH neurons also exist in mouse, but their relative abundance is different. Furthermore, divergences in the distribution of MCH axons were observed, in particular in the ventromedial hypothalamus. These differences suggest that rat and mouse MCH neurons are differentially involved in anatomical networks that control feeding and the sleep/wake cycle.