RESUMEN
El Clostridioides difficile (C. difficile) es una de las principales causas de infección asociada a la atención de salud con una elevada morbimortalidad, sobre todo en adultos mayores hospitalizados. El aumento en el uso de antibióticos ha ido de la mano con el incremento en el número de casos y de una mayor virulencia. Su presentación clínica va desde portadores asintomáticos hasta megacolon tóxico, escenarios que deben ser considerados al momento de realizar el estudio con exámenes de deposiciones (glutamato deshidrogenasa, toxinas A y B y técnicas de amplificación ácidos nucleares). Se han incorporado al arsenal terapéutico, con mayor nivel de evidencia, la fidaxomicina, trasplante microbiota fecal y recientemente nuevas terapias como anticuerpos monoclonales. Sin embargo, la gravedad de la infección, comorbilidad del paciente, presencia factores de recurrencia, el acceso y el costo económico de cada una de las opciones terapéuticas deben ser considerados. El objetivo de esta revisión es actualizar el manejo propuesto por las Sociedades Chilenas de Gastroenterología e Infectología publicadas el 2016 incorporando las últimas recomendaciones con respecto a prevención, diagnóstico y tratamiento de la infección por C. difficile.
Clostridioides difficile (C. difficile) is one of the leading causes of infection associated with health care with high morbidity and mortality, especially among hospitalized older adults. The increase in the use of antibiotics has been associated with a higher number of cases and greater virulence. Its clinical presentation ranges from asymptomatic carriers to toxic megacolon. Studies with stool tests (glutamate dehydrogenase, toxins A and B, and nuclear acid amplification techniques) should be considered in these cases. Fidaxomicin, fecal microbiota transplant, and new therapies such as monoclonal antibodies have been incorporated into the therapeutic arsenal, with a higher level of evidence. Nevertheless, the severity, patient comorbidity, recurrence risk factors, and the economic cost of each therapeutic option must be considered. This review aims to update the last guidelines proposed by the Chilean Societies of Gastroenterology and Infectious Diseases published in 2016, providing the latest recommendations regarding prevention, diagnosis, and treatment of C. difficile infection.
Asunto(s)
Humanos , Clostridioides difficile/patogenicidad , Infecciones por Clostridium/diagnóstico , Infecciones por Clostridium/terapia , Chile/epidemiología , Factores de Riesgo , Guías de Práctica Clínica como Asunto , Trasplante de Microbiota Fecal , Antibacterianos/uso terapéuticoRESUMEN
Clostridioides difficile (C. difficile) is one of the leading causes of infection associated with health care with high morbidity and mortality, especially among hospitalized older adults. The increase in the use of antibiotics has been associated with a higher number of cases and greater virulence. Its clinical presentation ranges from asymptomatic carriers to toxic megacolon. Studies with stool tests (glutamate dehydrogenase, toxins A and B, and nuclear acid amplification techniques) should be considered in these cases. Fidaxomicin, fecal microbiota transplant, and new therapies such as monoclonal antibodies have been incorporated into the therapeutic arsenal, with a higher level of evidence. Nevertheless, the severity, patient comorbidity, recurrence risk factors, and the economic cost of each therapeutic option must be considered. This review aims to update the last guidelines proposed by the Chilean Societies of Gastroenterology and Infectious Diseases published in 2016, providing the latest recommendations regarding prevention, diagnosis, and treatment of C. difficile infection.
Asunto(s)
Clostridioides difficile , Infecciones por Clostridium , Humanos , Infecciones por Clostridium/diagnóstico , Infecciones por Clostridium/terapia , Clostridioides difficile/patogenicidad , Antibacterianos/uso terapéutico , Factores de Riesgo , Guías de Práctica Clínica como Asunto , Trasplante de Microbiota Fecal , Chile/epidemiologíaRESUMEN
O objetivo deste estudo foi analisar a prevalência de Clostridioides difficile e suas toxinas (A/B) nas fezes de animais domésticos de um Hospital Veterinário Universitário de Teresina - PI. A detecção de C. difficile e suas toxinas foi realizada por meio de um ensaio imunoenzimático, denominado C. Diff Quik Chek Complete® (TECHLAB), capaz de detectar antígeno Glutamato Desidrogenase (GDH) e as toxinas A/B produzidas pelo bacilo, realizado em amostras fecais de cães (C. lupus) e e gatos (Felis catus) coletadas entre agosto de 2019 a setembro de 2020. Um total de 54 amostras fecais foram analisadas, das quais 16 foram positivas para C. difficile (29,63%). 68,75% (11/16) pertenciam a caninos, enquanto 31,25% (5/16) a felinos. Amostras diarreicas e não diarreicas foram utilizadas para o estudo e uma maior prevalência do bacilo pôde ser identificada em amostras diarreicas (33%). Nenhuma das amostras apresentou toxinas do patógeno. Os achados deste estudo evidenciam que C.difficile está presente no estado do Piauí. Foi possível identificá-lo em todas as espécies e em amostras diarreicas ou não, demonstrando que essa infecção pode se manifestar de formasintomática e assintomática, levantando a possibilidade de infecção cruzada entre o animal e seu tutor.
The aim of this study was to analyze the prevalence of Clostridioides difficile and its toxins (A/B) in the feces of domestic animals at a University Veterinary Hospital in Teresina - PI. The detection of C. difficile and its toxins was performed by an immunogenic enzyme, called C. Diff Quik Chek Complete® (TECHLAB), capable of detecting antigen glutamate dehydrogenase (GDH) and A/B toxins produced by this bacillus, performed in fecal samples of dogs (C. lupus) and cats (Felis catus) collected between August 2019 and September 2020.:54 stools were analyzed, of which 16 were positive for C. difficile (29.63%). 68.75% (11/16) belonged to canines, while 3.25% (5/16) to felines. Diarrheal and non-diarrheal diseases are used for the study and a higher prevalence of bacillus can be identified in diarrheal diseases (33%). None of the samples present pathogen toxins. The results of this study show that C. difficile is present in the state of Piauí. It can be identified in all species and in diarrheal or non-diarrheic samples, demonstrating that this infection can be symptomatic and asymptomatic, giving the possibility of cross-infection between the animal and its owner.
Asunto(s)
Animales , Gatos , Perros , Gatos/anomalías , Clostridioides difficile/patogenicidad , Técnicas para Inmunoenzimas/veterinaria , Infecciones por Clostridium/diagnóstico , Perros/anomalías , Heces/microbiología , Zoonosis Bacterianas/diagnósticoRESUMEN
Clostridioides difficile BI/NAP1/ribotype 027 is an epidemic hypervirulent strain found worldwide, including in Latin America. We examined the genomes and exoproteomes of two multilocus sequence type (MLST) clade 2 C. difficile strains considered hypervirulent: ICC-45 (ribotype SLO231/UK[CE]821), isolated in Brazil, and NAP1/027/ST01 (LIBA5756), isolated during a 2010 outbreak in Costa Rica. C. difficile isolates were cultured and extracellular proteins were analyzed using high-performance liquid chromatography-tandem mass spectrometry. Genomic analysis revealed that these isolates shared most of the gene composition. Only 83 and 290 NAP1/027 genes were considered singletons in ICC-45 and NAP1/027, respectively. Exoproteome analysis revealed 197 proteins, of which 192 were similar in both strains. Only five proteins were exclusive to the ICC-45 strain. These proteins were involved with catalytic and binding functions and indirectly interacted with proteins related to pathogenicity. Most proteins, including TcdA, TcdB, flagellin subunit, and cell surface protein, were overrepresented in the ICC-45 strain; 14 proteins, including mature S-layer protein, were present in higher proportions in LIBA5756. Data are available via ProteomeXchange with identifier PXD026218. These data show close similarity between the genome and proteins in the supernatant of two strains with hypervirulent features isolated in Latin America and underscore the importance of epidemiological surveillance of the transmission and emergence of new strains.
Asunto(s)
Clostridioides difficile/genética , Tipificación de Secuencias Multilocus , Clostridioides difficile/patogenicidad , Infecciones por Clostridium/epidemiología , Infecciones por Clostridium/microbiología , Brotes de Enfermedades , Humanos , América Latina/epidemiología , Tipificación de Secuencias Multilocus/métodos , Filogenia , Proteómica , RibotipificaciónRESUMEN
The role of gut microbiota in the establishment and development of Clostridioides difficile infection (CDI) has been widely discussed. Studies showed the impact of CDI on bacterial communities and the importance of some genera and species in recovering from and preventing infection. However, most studies have overlooked important components of the intestinal ecosystem, such as eukaryotes and archaea. We investigated the bacterial, archaea, and eukaryotic intestinal microbiota of patients with health-care-facility- or community-onset (HCFO and CO, respectively) diarrhea who were positive or negative for CDI. The CDI-positive groups (CO/+, HCFO/+) showed an increase in microorganisms belonging to Bacteroidetes, Firmicutes, Proteobacteria, Ascomycota, and Opalinata compared with the CDI-negative groups (CO/-, HCFO/-). Patients with intrahospital-acquired diarrhea (HCFO/+, HCFO/-) showed a marked decrease in bacteria beneficial to the intestine, and there was evidence of increased Archaea and Candida and Malassezia species compared with the CO groups (CO/+, CO/-). Characteristic microbiota biomarkers were established for each group. Finally, correlations between bacteria and eukaryotes indicated interactions among the different kingdoms making up the intestinal ecosystem. We showed the impact of CDI on microbiota and how it varies with where the infection is acquired, being intrahospital-acquired diarrhea one of the most influential factors in the modulation of bacterial, archaea, and eukaryotic populations. We also highlight interactions between the different kingdoms of the intestinal ecosystem, which need to be evaluated to improve our understanding of CDI pathophysiology.
Asunto(s)
Bacterias/clasificación , Infecciones por Clostridium/microbiología , Infecciones Comunitarias Adquiridas/microbiología , Infección Hospitalaria/microbiología , Diarrea/microbiología , Eucariontes/genética , Hongos/clasificación , Adulto , Anciano , Anciano de 80 o más Años , Bacterias/genética , Bacterias/aislamiento & purificación , Clostridioides difficile/patogenicidad , Eucariontes/clasificación , Eucariontes/aislamiento & purificación , Femenino , Hongos/genética , Hongos/aislamiento & purificación , Microbioma Gastrointestinal , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Masculino , Persona de Mediana Edad , Filogenia , ARN Ribosómico 16S/genética , ARN Ribosómico 18S/genética , Análisis de Secuencia de ADN , Adulto JovenRESUMEN
Clostridioides (Clostridium) difficile is the main causative agent of antimicrobial-related diarrhea in humans and a major pathogen-associated enteric disorder in foals and adult horses. Moreover, studies have suggested that animals are a possible reservoir of toxigenic C. difficile strains for humans. Despite this known importance, the epidemiology of C. difficile infection (CDI) in equine is still largely unknown. Therefore, this study described six cases of equine CDI occurring in Minas Gerais, Brazil, including the characterization of the isolates. All but one equine included in this research developed CDI after antimicrobial therapy, three of which occurred during hospitalization. Coinfection with Salmonella Heidelberg and S. Infantis was detected in three cases, making the antimicrobial treatment challenging. All animals recovered after metronidazole administration. All C. difficile isolates were susceptible to metronidazole and vancomycin, while three were resistant to moxifloxacin and two were resistant to clindamycin. The isolates were classified as RT126 (n = 4), RT078 (n = 1), and RT014/020 (n = 1), all previously reported infecting humans and animals worldwide.(AU)
Clostridioides (Clostridium) difficile é o principal agente envolvido em diarreias associadas ao uso de antimicrobianos em seres humanos e um enteropatógeno de grande relevância em quadros de diarreia em potros e equinos adultos. Em adição, estudos tem sugerido que animais são possíveis reservatórios de estirpes toxigênicas de C. difficile para humanos. Apesar da importância na saúde animal e humana, a epidemiologia da infecção por C. difficile (ICD) é ainda pouco conhecida. Dessa forma, o presente estudo tem como objetivo caracterizar seis casos de diarreia por C. difficile ocorridos em Minas Gerais, Brasil. Com exceção de um animal, todos os equinos incluídos no presente estudo desenvolveram ICD após antibioticoterapia, três dos quais durante a hospitalização. Coinfecção por Salmonella Heidelberg e S. Infantis foi detectada em três casos, tornando o tratamento antimicrobiano desafiador. Todos os animais recuperaram após administração de metronidazol. Os isolados obtidos no presente estudo foram sensíveis a metronidazol e vancomicina, porém três estirpes foram resistentes a moxifloxacina e duas a clindamicina. Os isolados foram classificados como ribotipos 126 (n=4), 078 (n=1) e 014/020 (n=1), todos previamente relatados em seres humanos com ICD no Brasil e em outros países.(AU)
Asunto(s)
Animales , Equidae , Enterocolitis Seudomembranosa/veterinaria , Clostridioides difficile/genética , Clostridioides difficile/patogenicidad , Infecciones por Clostridium/diagnóstico , Infecciones por Clostridium/tratamiento farmacológico , Infecciones por Clostridium/microbiología , Infecciones por Clostridium/epidemiología , Infecciones por Clostridium/veterinariaRESUMEN
Clostridioides (Clostridium) difficile is the main causative agent of antimicrobial-related diarrhea in humans and a major pathogen-associated enteric disorder in foals and adult horses. Moreover, studies have suggested that animals are a possible reservoir of toxigenic C. difficile strains for humans. Despite this known importance, the epidemiology of C. difficile infection (CDI) in equine is still largely unknown. Therefore, this study described six cases of equine CDI occurring in Minas Gerais, Brazil, including the characterization of the isolates. All but one equine included in this research developed CDI after antimicrobial therapy, three of which occurred during hospitalization. Coinfection with Salmonella Heidelberg and S. Infantis was detected in three cases, making the antimicrobial treatment challenging. All animals recovered after metronidazole administration. All C. difficile isolates were susceptible to metronidazole and vancomycin, while three were resistant to moxifloxacin and two were resistant to clindamycin. The isolates were classified as RT126 (n = 4), RT078 (n = 1), and RT014/020 (n = 1), all previously reported infecting humans and animals worldwide.
Clostridioides (Clostridium) difficile é o principal agente envolvido em diarreias associadas ao uso de antimicrobianos em seres humanos e um enteropatógeno de grande relevância em quadros de diarreia em potros e equinos adultos. Em adição, estudos tem sugerido que animais são possíveis reservatórios de estirpes toxigênicas de C. difficile para humanos. Apesar da importância na saúde animal e humana, a epidemiologia da infecção por C. difficile (ICD) é ainda pouco conhecida. Dessa forma, o presente estudo tem como objetivo caracterizar seis casos de diarreia por C. difficile ocorridos em Minas Gerais, Brasil. Com exceção de um animal, todos os equinos incluídos no presente estudo desenvolveram ICD após antibioticoterapia, três dos quais durante a hospitalização. Coinfecção por Salmonella Heidelberg e S. Infantis foi detectada em três casos, tornando o tratamento antimicrobiano desafiador. Todos os animais recuperaram após administração de metronidazol. Os isolados obtidos no presente estudo foram sensíveis a metronidazol e vancomicina, porém três estirpes foram resistentes a moxifloxacina e duas a clindamicina. Os isolados foram classificados como ribotipos 126 (n=4), 078 (n=1) e 014/020 (n=1), todos previamente relatados em seres humanos com ICD no Brasil e em outros países.
Asunto(s)
Animales , Clostridioides difficile/genética , Clostridioides difficile/patogenicidad , Enterocolitis Seudomembranosa/veterinaria , Equidae , Infecciones por Clostridium/diagnóstico , Infecciones por Clostridium/epidemiología , Infecciones por Clostridium/microbiología , Infecciones por Clostridium/tratamiento farmacológico , Infecciones por Clostridium/veterinariaRESUMEN
Clostridioides difficile is an obligately anaerobic, spore-forming, Gram-positive pathogenic bacterium that is considered the leading cause of nosocomial diarrhea worldwide. Recent studies have attempted to understand the biology of the outermost layer of C. difficile spores, the exosporium, which is believed to contribute to early interactions with the host. The fundamental role of the cysteine-rich proteins CdeC and CdeM has been described. However, the molecular details behind the mechanism of exosporium assembly are missing. The underlying mechanisms that govern exosporium assembly in C. difficile remain poorly studied, in part due to difficulties in obtaining pure soluble recombinant proteins of the C. difficile exosporium. In this work, we observed that CdeC was able to form organized inclusion bodies (IBs) in Escherichia coli filled with lamella-like structures separated by an interspace of 5 to 15 nm; however, CdeC expression in an E. coli strain with a more oxidative environment led to the loss of the lamella-like organization of CdeC IBs. Additionally, dithiothreitol (DTT) treatment of CdeC inclusion bodies released monomeric soluble forms of CdeC. Deletions in different portions of CdeC did not affect CdeC's ability to aggregate and form oligomers stable under denaturation conditions but affected CdeC's self-assembly properties. Overall, these observations have important implications in further studies elucidating the role of CdeC in the exosporium assembly of C. difficile spores.IMPORTANCE The endospore of Clostridioides difficile is the vehicle for transmission and persistence of the pathogen, and, specifically, the exosporium is the first contact between the host and the spore. The underlying mechanisms that govern exosporium assembly in C. difficile remain understudied, in part due to difficulties in obtaining pure soluble recombinant proteins of the C. difficile exosporium. Understanding the exosporium assembly's molecular bases may be essential to developing new therapies against C. difficile infection.
Asunto(s)
Proteínas Bacterianas/metabolismo , Clostridioides difficile/patogenicidad , Cuerpos de Inclusión/metabolismo , Esporas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Pared Celular/química , Pared Celular/metabolismo , Clostridioides difficile/química , Clostridioides difficile/metabolismo , Cisteína/química , Cisteína/metabolismo , Escherichia coli/metabolismo , Esporas Bacterianas/químicaRESUMEN
La diarrea clostridial es una enfermedad aguda con compromiso colónico que puede poner en riesgo la vida de un paciente. Su agente etiológico es el Clostridium difficile y se ha asociado al uso indiscriminado y por largo plazo de antibióticos de amplio espectro. Su cuadro clínico es variable, puede ir desde un cuadro de diarrea hasta la perforación colónica, que puede determinar la realización de una colectomía de urgencia o incluso provocar la muerte del enfermo. El diagnóstico de certeza se realiza mediante la detección de la toxina clostridial en materia fecal, por técnicas de inmunoensayo enzimático. La terapéutica se realiza con metronidazol o vancomicina por vía oral. El tratamiento quirúrgico está indicado ante la presencia de megacolon tóxico o perforación intestinal, y en aquellos pacientes con toxicidad sistémica con fracaso de la terapéutica médica. (AU)
Clostridial diarrhea is an acute disease with colonic involvement that can be life-threatening for a patient. Its etiologic agent is the Clostridium difficile and it has been associated with the indiscriminate and long-term use of broad-spectrum antibiotics. Its clinical picture varies from a picture of diarrhea to colonic perforation that can determine the performance of an emergency colectomy or even the death of the patient. The certainty diagnosis is carried out by detecting clostridial toxin in fecal matter by enzyme immunoassay techniques. The therapy is carried out with metronidazole or vancomycin orally. Surgical treatment is indicated in the presence of toxic mega colon, intestinal perforation or in those patients with systemic toxicity with failure of medical therapy. (AU)
Asunto(s)
Humanos , Enterocolitis Seudomembranosa/inducido químicamente , Clostridioides difficile/patogenicidad , Antibacterianos/efectos adversos , Enterocolitis Seudomembranosa/diagnóstico , Enterocolitis Seudomembranosa/terapia , Diagnóstico por Imagen , Metronidazol/administración & dosificaciónRESUMEN
In piglets, Clostridioides (C.) difficile infection presents mostly subclinical manifestation. As this agent became important in veterinary medicine due to a hypothesis of zoonosis, the objective of this study was to evaluate the transmission of C. difficile by nose-to-nose contact in young piglets. Six 20-day-old piglets were separated into three groups (infected, sentinel and control), and distributed in different isolation cabinets which allowed nose-to-nose contact only between infected and sentinel groups. The challenged group received an inoculum 106 CFU/mL of C. difficile 096 by oropharyngeal route. Rectal swab samples were daily collected for microbiological and molecular analysis. Euthanasia of all piglets was performed 18 days after challenge to evaluate anatomical, histological and microbiological lesions of the organs of these animals. The challenged and sentinel groups showed clinical signs of infection and genes encoding TcdB were detected by conventional PCR in both groups, confirming the transmission of the pathogen from the challenged to the sentinel piglets. At necropsy, tonsil, liver, spleen, mesenteric lymph nodes, ileocolic lymph nodes, jejunum, ileum, proximal colon, distal colon and cecum were collected for microbiological analysis; lesions were observed varying in degree and intensity. This study demonstrated a novel route of transmission of C. difficile between young piglets, which was proven to occur by nose-to-nose contact.
Asunto(s)
Clostridioides difficile/patogenicidad , Infecciones por Clostridium/transmisión , Infecciones por Clostridium/veterinaria , Nariz/microbiología , Enfermedades de los Porcinos/transmisión , Destete , Factores de Edad , Animales , Heces/microbiología , Recto/microbiología , Porcinos/microbiología , Enfermedades de los Porcinos/microbiologíaRESUMEN
Antibiotic-induced dysbiosis is a key factor predisposing intestinal infection by Clostridium difficile. Here, we show that interventions that restore butyrate intestinal levels mitigate clinical and pathological features of C. difficile-induced colitis. Butyrate has no effect on C. difficile colonization or toxin production. However, it attenuates intestinal inflammation and improves intestinal barrier function in infected mice, as shown by reduced intestinal epithelial permeability and bacterial translocation, effects associated with the increased expression of components of intestinal epithelial cell tight junctions. Activation of the transcription factor HIF-1 in intestinal epithelial cells exerts a protective effect in C. difficile-induced colitis, and it is required for butyrate effects. We conclude that butyrate protects intestinal epithelial cells from damage caused by C. difficile toxins via the stabilization of HIF-1, mitigating local inflammatory response and systemic consequences of the infection.
Asunto(s)
Butiratos/administración & dosificación , Clostridioides difficile/patogenicidad , Colitis/prevención & control , Factor 1 Inducible por Hipoxia/metabolismo , Administración Oral , Animales , Antibacterianos/farmacología , Butiratos/farmacología , Clostridioides difficile/metabolismo , Colitis/etiología , Colitis/microbiología , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Ácidos Grasos Volátiles/metabolismo , Humanos , Insulina/administración & dosificación , Mucosa Intestinal/citología , Mucosa Intestinal/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Microbiota/efectos de los fármacos , Permeabilidad/efectos de los fármacos , Uniones Estrechas/metabolismo , Toxinas Biológicas/toxicidad , Triglicéridos/administración & dosificaciónRESUMEN
Neonatal porcine diarrhea (NPD) is a current problem on pig farms and is caused by several enteropathogens. Among them, Clostridioides difficile stands out due to its importance in piglets and zoonotic potential. A non-toxigenic strain of C. difficile (NTCD), named Z31, was previously tested in hamster and piglet experimental models as a strategy to prevent C. difficile infection (CDI). To evaluate the capacity of the strain Z31 to prevent CDI and NPD in one-day-old piglets on a commercial farm, 90 piglets from 16 litters received 1 × 106 spores of Z31 while 84 animals from the same litters served as controls. Animals were clinically evaluated, and fecal samples were collected 24 h after administration and submitted to A/B toxin detection and isolation of C. difficile. Stool samples were also submitted to rotavirus, Escherichia coli, and Clostridium perfringens detection. Administration of Z31 reduced the incidence of CDI in treated animals (7.8%) when compared to the control group (25.0%; P = 0.003). In animals that developed CDI, the intensity of diarrhea was lower in those that received Z31 than in the control group. Neonatal porcine diarrhea was reduced in treated animals when compared to untreated animals (P < 0.001). The present study suggests that Z31 can potentially be used to prevent CDI in piglets on commercial farms.
Asunto(s)
Clostridioides difficile/fisiología , Clostridioides difficile/patogenicidad , Infecciones por Clostridium/prevención & control , Diarrea/veterinaria , Enfermedades de los Porcinos/microbiología , Animales , Animales Recién Nacidos , Derrame de Bacterias , Infecciones por Clostridium/microbiología , Clostridium perfringens/aislamiento & purificación , Diarrea/microbiología , Diarrea/prevención & control , Escherichia coli Enterotoxigénica/aislamiento & purificación , Granjas , Heces/microbiología , Porcinos , Enfermedades de los Porcinos/prevención & controlRESUMEN
The population structure of Clostridium difficile currently comprises eight major genomic clades. For the highly divergent C-I clade, only two toxigenic strains have been reported, which lack the tcdA and tcdC genes and carry a complete locus for the binary toxin (CDT) next to an atypical TcdB monotoxin pathogenicity locus (PaLoc). As part of a routine surveillance of C. difficile in stool samples from diarrheic human patients, we discovered three isolates that consistently gave negative results in a PCR-based screening for tcdC. Through phenotypic assays, whole-genome sequencing, experiments in cell cultures, and infection biomodels we show that these three isolates (i) escape common laboratory diagnostic procedures, (ii) represent new ribotypes, PFGE-types, and sequence types within the Clade C-I, (iii) carry chromosomal or plasmidal TcdBs that induce classical or variant cytopathic effects (CPE), and (iv) cause different levels of cytotoxicity and hamster mortality rates. These results show that new strains of C. difficile can be detected by more refined techniques and raise questions on the origin, evolution, and distribution of the toxin loci of C. difficile and the mechanisms by which this emerging pathogen causes disease.
Asunto(s)
Toxinas Bacterianas/genética , Cromosomas Bacterianos/genética , Clostridioides difficile/genética , Clostridioides difficile/patogenicidad , Virulencia/genética , Proteínas Bacterianas/genética , Línea Celular Tumoral , Pruebas Diagnósticas de Rutina/métodos , Enterotoxinas/genética , Células HeLa , Humanos , Filogenia , Ribotipificación/métodos , Secuenciación Completa del Genoma/métodosRESUMEN
Clostridium difficile is a Gram-positive spore-former bacterium and the leading cause of nosocomial antibiotic-associated diarrhea that can culminate in fatal colitis. During the infection, C. difficile produces metabolically dormant spores, which persist in the host and can cause recurrence of the infection. The surface of C. difficile spores seems to be the key in spore-host interactions and persistence. The proteome of the outermost exosporium layer of C. difficile spores has been determined, identifying two cysteine-rich exosporium proteins, CdeC and CdeM. In this work, we explore the contribution of both cysteine-rich proteins in exosporium integrity, spore biology and pathogenesis. Using targeted mutagenesis coupled with transmission electron microscopy we demonstrate that both cysteine rich proteins, CdeC and CdeM, are morphogenetic factors of the exosporium layer of C. difficile spores. Notably, cdeC, but not cdeM spores, exhibited defective spore coat, and were more sensitive to ethanol, heat and phagocytic cells. In a healthy colonic mucosa (mouse ileal loop assay), cdeC and cdeM spore adherence was lower than that of wild-type spores; while in a mouse model of recurrence of the disease, cdeC mutant exhibited an increased infection and persistence during recurrence. In a competitive infection mouse model, cdeC mutant had increased fitness over wild-type. Through complementation analysis with FLAG fusion of known exosporium and coat proteins, we demonstrate that CdeC and CdeM are required for the recruitment of several exosporium proteins to the surface of C. difficile spores. CdeC appears to be conserved exclusively in related Peptostreptococcaeace family members, while CdeM is unique to C. difficile. Our results sheds light on how CdeC and CdeM affect the biology of C. difficile spores and the assembly of the exosporium layer and, demonstrate that CdeC affect C. difficile pathogenesis.
Asunto(s)
Proteínas Bacterianas/metabolismo , Clostridioides difficile/patogenicidad , Infecciones por Clostridium/metabolismo , Esporas Bacterianas/metabolismo , Animales , Proteínas Bacterianas/química , Pared Celular/química , Pared Celular/metabolismo , Clostridioides difficile/química , Clostridioides difficile/metabolismo , Cisteína/química , Cisteína/metabolismo , Interacciones Huésped-Patógeno/fisiología , Ratones , Esporas Bacterianas/químicaRESUMEN
PURPOSE: Clostridium difficile infections are the leading cause of diarrhea associated with the use of antibiotics. During infection, C. difficile initiates a sporulation cycle leading to the persistence of C. difficile spores in the host and disease dissemination. The development of vaccine and passive immunization therapies against C. difficile has focused on toxins A and B. In this study, an immunoproteome-based approach to identify immunogenic proteins located on the outer layers of C. difficile spores as potential candidates for the development of immunotherapy and/or diagnostic methods against this devastating infection is used. EXPERIMENTAL DESIGN: To identify potential immunogenic proteins on the surface of C. difficile R20291, spore coat/exosporium extracts are separated by 2D electrophoresis (2-DE) and analyzed for reactivity against C. difficile spore-specific goat sera. Finally, the selected spots are in-gel digested with chymotrypsin, peptides generated are separated by nanoUPLC followed by MS/MS using Quad-TOF-MS, corroborated by Ultimate 3000RS-nano-UHPLC coupled to Q-Exactive-Plus-Orbitrap MS. RESULTS: The analysis identify five immunoreactive proteins: spore coat proteins CotE, CotA, and CotCB; exosporium protein CdeC; and a cytosolic methyltransferase. CONCLUSION: This data provides a list of spore surface protein candidates as antigens for vaccine development against C. difficile infections.
Asunto(s)
Proteínas Bacterianas/aislamiento & purificación , Clostridioides difficile/genética , Enterocolitis Seudomembranosa/diagnóstico , Proteínas de la Membrana/genética , Proteínas Bacterianas/genética , Pared Celular/genética , Clostridioides difficile/patogenicidad , Enterocolitis Seudomembranosa/genética , Enterocolitis Seudomembranosa/microbiología , Humanos , Proteínas de la Membrana/aislamiento & purificación , Esporas Bacterianas/genética , Espectrometría de Masas en TándemRESUMEN
BACKGROUND AND AIM: Clostridium difficile is a major cause of health care-associated infection, but disagreement between diagnostic tests is an ongoing barrier to clinical decision-making. Conventional enzyme immunoassay (EIA) for toxin detection is currently the most frequently used technique for C. difficile infection (CDI) diagnosis, but its low sensitivity makes the development of an alternative strategy necessary for improving the diagnosis in developing countries. METHODS: Between years 2011 and 2015, 154 stool samples from patients with antibiotic-associated diarrhea were examined by toxigenic culture and EIA for the diagnosis of CDI. In the year 2015, when glutamate dehydrogenase (GDH) test was first available in Brazil, 53 of those fecal specimens were also tested by the C. diff Quik Chek Complete rapid immunoassay. At this time, we prospectively assessed the impact of this test on CDI treatment rates before and after it was introduced in clinical practice. RESULTS: The GDH component of C. diff Quik Chek Complete test had a sensitivity of 100% and specificity of 95.1% compared with toxigenic culture, with 89.8% concordance. The Tox A/B II EIA and the toxin portion of C. diff Quik Chek Complete yielded sensitivities between values of 50-58.3%, with 100% specificities. The introduction of GDH test increased the number of treated patients with CDI from 57.7% to 100%. CONCLUSIONS: Glutamate dehydrogenase test is a reliable method for the diagnosis of CDI and greatly increases the number of properly treated patients with CDI. Therefore, this exam should be considered the mainstay for the laboratory diagnosis of CDI in developing countries.
Asunto(s)
Antibacterianos/efectos adversos , Proteínas Bacterianas/análisis , Toxinas Bacterianas/análisis , Clostridioides difficile/patogenicidad , Infecciones por Clostridium/diagnóstico , Diarrea/etiología , Diarrea/microbiología , Enterotoxinas/análisis , Glutamato Deshidrogenasa/análisis , Técnicas para Inmunoenzimas/métodos , Colorantes Azulados , Biomarcadores/análisis , Brasil , Clostridioides difficile/enzimología , Clostridioides difficile/metabolismo , Infecciones por Clostridium/microbiología , Heces/microbiología , Hospitales Universitarios , Humanos , Azul de Metileno , Estudios Prospectivos , Sensibilidad y Especificidad , XantenosRESUMEN
Resumen Introducción. Clostridium difficile es el principal responsable de la diarrea asociada al uso de antibióticos. En Colombia y en Latinoamérica, el conocimiento sobre el comportamiento epidemiológico de la infección por C. difficile todavía es limitado. Objetivo. Describir las características de una serie de pacientes con infección por C.difficile . Materiales y métodos. Se hizo un estudio descriptivo de una serie de casos de pacientes con infección por C. difficile atendidos en la Fundación Clínica Shaio, entre enero de 2012 y noviembre de 2015. Resultados. Se estudiaron 36 pacientes con una edad promedio de 65 años. Se determinaron los siguientes factores relacionados con la infección por C. difficile: uso previo de antimicrobianos (94,4 %), hospitalización en los últimos tres meses (66,7 %) y uso de inhibidores de la bomba de protones (50 %). Las comorbilidades más comunes fueron la enfermedad renal crónica (41,7 %) y la diabetes mellitus (30,6 %). Los síntomas más frecuentes fueron más de tres deposiciones diarreicas (97,1 %) y dolor abdominal (42,9 %). En cuanto a la gravedad de los casos, 44,4 % se clasificó como leve a moderado, 38,9 % como grave, y 11,1 % como complicado o grave. El método de diagnóstico más utilizado (63,8% de los pacientes) fue la identificación de la toxina mediante reacción en cadena de la polimerasa (PCR). La mortalidad global durante la hospitalización fue de 8 %. Se identificaron cuatro cepas del serotipo NAP1/027 y nueve muestras fueron positivas para la toxina binaria. Conclusión. La infección por C. difficile debe sospecharse en pacientes con deposiciones diarreicas y factores asociados tradicionalmente a esta enfermedad. Se reportó la circulación de cepas hipervirulentas del serotipo NAP1/027 en Colombia, lo cual debe enfrentarse con la vigilancia epidemiológica y el diagnóstico temprano.
Abstract Introduction: Clostridium difficile is the main pathogen related to healthcare-associated diarrhea and it is the cause of 20 to 30% of diarrhea cases caused by antibiotics. In Colombia and Latin America, the knowledge about the epidemiological behavior of this infection is limited. Objective: To describe the characteristics of a series of patients with C. difficile infection. Materials and methods: We performed a descriptive case series study of patients with C. difficile infection hospitalized in the Fundación Clínica Shaio from January, 2012, to November, 2015. Results: We analyzed 36 patients. The average age was 65 years. The risk factors associated with the infection were: previous use of antibiotics (94.4%), prior hospitalization in the last three months (66.7%) and use of proton pump inhibitors (50%). The most common comorbidities were chronic kidney disease (41.7%) and diabetes mellitus (30.6%). The most frequent symptoms were more than three loose stools per day (97.1%) and abdominal pain (42.9%). According to the severity of the disease, 44.4% of cases were classified as mild to moderate, 38.9% as severe, and 11.1% as complicated or severe. The detection of the toxin by PCR (GeneXpert) was the most common diagnostic procedure (63.8%). Global mortality during hospitalization was 8%. We identified four strains with serotype NAP1/027 and nine samples positive for binary toxin. Conclusion: Clostridium difficile infection should be suspected in patients with diarrhea and traditional risk factors associated with this disease. We report the circulation of the hypervirulent strain serotype NAP1/027 in Colombia, which should be countered with epidemiological surveillance and a prompt diagnosis.
Asunto(s)
Anciano , Anciano de 80 o más Años , Femenino , Humanos , Masculino , Persona de Mediana Edad , Infección Hospitalaria/microbiología , Clostridioides difficile/aislamiento & purificación , Infecciones por Clostridium/microbiología , Proteínas Bacterianas/análisis , Toxinas Bacterianas/análisis , Virulencia , Serotipificación , Dolor Abdominal/etiología , Comorbilidad , Infección Hospitalaria/epidemiología , Factores de Riesgo , Clostridioides difficile/clasificación , Clostridioides difficile/patogenicidad , Infecciones por Clostridium/epidemiología , Colombia/epidemiología , Diabetes Mellitus/epidemiología , Diarrea/microbiología , Diarrea/epidemiología , Insuficiencia Renal Crónica/epidemiología , Inhibidores de la Bomba de Protones/efectos adversos , Inhibidores de la Bomba de Protones/uso terapéutico , Hospitalización , Tiempo de Internación/estadística & datos numéricos , Antibacterianos/efectos adversos , Antibacterianos/uso terapéuticoRESUMEN
BACKGROUND: The diagnosis of Clostridium difficile infection (CDI) increases concern that asymptomatic carriers of toxigenic C. difficile may be diagnosed with CDI. METHODS: A matched case control study was conducted in inpatients in a tertiary care center. The first 50 patients with diarrhea and a positive polymerase chain reaction (PCR) test beginning February 1, 2015, were identified as cases. Control patients were hospitalized patients receiving antibiotics, but with no diarrhea, housed in a room as close as possible to each case during the same admission time. A convenience sample of healthcare workers who cared for C. difficile infected patients was also tested. RESULTS: We found two positive PCR results for C. difficile in controls (4.1%). None of these healthcare workers were positive for C. difficile by PCR. There was no difference between groups with respect to overall antibiotic use before the requested PCR for Clostridium difficile (p = 0.359). The majority of cases had a high proportion of gastrointestinal disorders (71.4%) compared with control (8.2%), p < 0.001. Patients with neoplasia had a higher chance of being identified as cases (p = 0.041). CONCLUSIONS: PCR should not be the only diagnostic tool but should be complementary to other methods and to the medical history.
Asunto(s)
Clostridioides difficile/genética , Infecciones por Clostridium/microbiología , Diarrea/microbiología , Adulto , Anciano , Anciano de 80 o más Años , Antibacterianos/uso terapéutico , Estudios de Casos y Controles , Clostridioides difficile/aislamiento & purificación , Clostridioides difficile/patogenicidad , Infecciones por Clostridium/genética , Diarrea/genética , Diarrea/patología , Femenino , Heterocigoto , Humanos , Masculino , Persona de Mediana Edad , Factores de Riesgo , Centros de Atención TerciariaRESUMEN
Clostridium difficile, the main cause of diarrhea in hospitalized patients, produces toxins A (TcdA) and B (TcdB), which affect intestinal epithelial cell survival, proliferation, and migration and induce an intense inflammatory response. Transforming growth factor ß (TGF-ß) is a pleiotropic cytokine affecting enterocyte and immune/inflammatory responses. However, it has been shown that exposure of intestinal epithelium to a low concentration of TcdA induces the release of TGF-ß1, which has a protective effect on epithelial resistance and a TcdA/TGF-ß signaling pathway interaction. The activation of this pathway in vivo has not been elucidated. The aim of this study was to investigate the role of the TGF-ß1 pathway in TcdA-induced damage in a rat intestinal epithelial cell line (IEC-6) and in a mouse model of an ileal loop. TcdA increased the expression of TGF-ß1 and its receptor, TßRII, in vitro and in vivo TcdA induced nuclear translocation of the transcription factors SMAD2/3, a hallmark of TGF-ß1 pathway activation, both in IEC cells and in mouse ileal tissue. The addition of recombinant TGF-ß1 (rTGF-ß) prevented TcdA-induced apoptosis/necrosis and restored proliferation and repair activity in IEC-6 cells in the presence of TcdA. Together, these data show that TcdA induces TGF-ß1 signaling pathway activation and suggest that this pathway might play a protective role against the effect of C. difficile-toxin.