ABSTRACT
Clostridioides difficile (C. difficile) is widely distributed in the intestinal tract of humans, animals, and in the environment. It is the most common cause of diarrhea associated with the use of antimicrobials in humans and among the most common healthcare-associated infections worldwide. Its pathogenesis is mainly due to the production of toxin A (TcdA), toxin B (TcdB), and a binary toxin (CDT), whose genetic variants may be associated with disease severity. We studied genetic diversity in 39 C. difficile isolates from adults and children attended at two Mexican hospitals, using different gene and genome typing methods and investigated their association with in vitro expression of toxins. Whole-genome sequencing in 39 toxigenic C. difficile isolates were used for multilocus sequence typing, tcdA, and tcdB typing sequence type, and phylogenetic analysis. Strains were grown in broth media, and expression of toxin genes was measured by real-time PCR and cytotoxicity in cell-culture assays. Clustering of strains by genome-wide phylogeny matched clade classification, forming different subclusters within each clade. The toxin profile tcdA+/tcdB+/cdt+ and clade 2/ST1 were the most prevalent among isolates from children and adults. Isolates presented two TcdA and three TcdB subtypes, of which TcdA2 and TcdB2 were more prevalent. Prevalent clades and toxin subtypes in strains from children differed from those in adult strains. Toxin gene expression or cytotoxicity was not associated with genotyping or toxin subtypes. In conclusion, genomic and phenotypic analysis shows high diversity among C. difficile isolates from patients with healthcare-associated diarrhea. IMPORTANCE: Clostridioides difficile is a toxin-producing bacterial pathogen recognized as the most common cause of diarrhea acquired primarily in healthcare settings. This bacterial species is diverse; its global population has been divided into five different clades using multilocus sequence typing, and strains may express different toxin subtypes that may be related to the clades and, importantly, to the severity and progression of disease. Genotyping of children strains differed from adults suggesting toxins might present a reduced toxicity. We studied extensively cytotoxicity, expression of toxins, whole genome phylogeny, and toxin typing in clinical C. difficile isolates. Most isolates presented a tcdA+/ tcdB+/cdt+ pattern, with high diversity in cytotoxicity and clade 2/ST1 was the most prevalent. However, they all had the same TcdA2/TcdB2 toxin subtype. Advances in genomics and bioinformatics tools offer the opportunity to understand the virulence of C. difficile better and find markers for better clinical use.
Subject(s)
Bacterial Toxins , Clostridioides difficile , Clostridium Infections , Cross Infection , Diarrhea , Genetic Variation , Multilocus Sequence Typing , Phylogeny , Humans , Clostridioides difficile/genetics , Clostridioides difficile/classification , Clostridioides difficile/isolation & purification , Diarrhea/microbiology , Diarrhea/epidemiology , Mexico/epidemiology , Child , Bacterial Toxins/genetics , Adult , Clostridium Infections/microbiology , Clostridium Infections/epidemiology , Cross Infection/microbiology , Cross Infection/epidemiology , Bacterial Proteins/genetics , Enterotoxins/genetics , Male , Child, Preschool , Female , Prevalence , Adolescent , Whole Genome Sequencing , Phenotype , Genome, Bacterial/genetics , Infant , Middle Aged , GenomicsABSTRACT
Bacillus cereus is an aerobic and facultatively anaerobic, spore-forming bacterium, and it is found naturally in soil and poses a risk factor for the contamination of food and foodstuffs including cereals, vegetables, spices, ready-to-eat (RTE) foods, meats, milk, and dairy products. This study determined the prevalence of B. cereus in raw poultry meat, raw cow's milk, cheese, spices, and RTE foods in Hatay province. The study also analysed the psychrotrophic properties, toxigenic characteristics, and pulsed-field gel electrophoresis (PFGE) profiles of the isolates. The levels of contamination with B. cereus determined for cheese, raw milk, RTE foods, spices, and raw poultry meat were 16.6%, 34.2%, 42.8%, 49%, and 55.5%, respectively. B. cereus was isolated from 84 (42%) of the 200 samples analysed and the 84 isolates were verified by PCR analysis targeting the haemolysin gene specific for B. cereus. Of the total isolates, 64 (76.1%) were psychrotrophic. The toxin gene profiling of B. cereus isolates was determined by amplifying the four genes nhe, hbl, cytK, and ces. The nhe and cytK genes were most frequently detected in the isolates, while the hbl and ces genes were not found. In addition, a high genetic relationship between the isolates was detected at a 92% similarity level by PFGE analysis. In conclusion, the occurrence of both psychrotrophic and toxigenic B. cereus strains in this study indicated a potential risk for food spoilage and food poisoning.
Bacillus cereus é uma bactéria formadora de esporos aeróbica e facultativamente anaeróbica, encontrada naturalmente no solo e representa um fator de risco para a contaminação de alimentos e alimentos, incluindo cereais, vegetais, especiarias, alimentos prontos para comer (RTE), carnes, leite e laticínios. Este estudo teve como objetivo determinar a prevalência de B. cereus em carne crua de aves, leite de vaca cru, queijo, especiarias e alimentos RTE na província de Hatay. O estudo também analisou as propriedades psicrotróficas, características toxigênicas e perfis de PFGE dos isolados. Os níveis de contaminação com B. cereus determinados para queijo, leite cru, alimentos RTE, especiarias e carne de frango crua foram de 16,6%, 34,2%, 42,8%, 49% e 55,5%, respectivamente. B. cereus foi isolado de 84 (42%) das 200 amostras analisadas e os 84 isolados foram verificados por análise de PCR visando o gene da hemolisina específico para B. cereus. Do total de isolados, 64 (76,1%) eram psicrotróficos. O perfil do gene da toxina de isolados de B. cereus foi determinado pela amplificação dos quatro genes nhe, hbl, cytK e ces. Os genes nhe e cytK foram detectados com maior frequência nos isolados, enquanto os genes hbl e ces não foram encontrados. Além disso, uma alta relação genética entre os isolados foi detectada em um nível de similaridade de 92% pela análise de PFGE. Em conclusão, a ocorrência de cepas psicrotróficas e toxigênicas de B. cereus neste estudo indica um risco potencial de deterioração e intoxicação alimentar.
Subject(s)
Bacillus cereus/isolation & purification , Bacillus cereus/classification , Bacillus cereus/genetics , Foodborne Diseases , Polymerase Chain Reaction , Bacterial Typing TechniquesABSTRACT
Clostridium perfringens type A has been incriminated as the etiologic agent in jejunal hemorrhage syndrome (JHS), which is a disease that affects dairy cattle. Although this microorganism is considered an important enteropathogen the pathogenesis of JHS is still not clear, and there have been no reports of its occurrence in Brazil so far. The aim of this study was to describe the occurrence of JHS by infection with a C. perfringens type A strain carrying the beta-2 toxin gene in a zebu cow in Brazil, for the first time.
Clostridium perfringens tipo A tem sido considerado agente etiológico da síndrome do jejuno hemorrágico (SJH), que é uma doença que afeta comumente os rebanhos de gado. Embora este microrganismo seja considerado um importante enteropatógeno, a patogênese da SJH ainda não foi elucidada, e não havia sido reportada no Brasil até então. O alvo deste estudo foi descrever pela primeira vez a ocorrência da SJH causada por C. perfringens tipo A, carreador do gene da toxina beta-2, em um zebuíno no Brasil.
ABSTRACT
Clostridium perfringenstype A has been incriminated as the etiologic agent in jejunal hemorrhage syndrome (JHS), which is a disease that affects dairy cattle. Although this microorganism is considered an important enteropathogen the pathogenesis of JHS is still not clear, and there have been no reports of its occurrence in Brazil so far. The aim of this study was to describe the occurrence of JHS by infection with a C. perfringenstype A strain carrying the beta-2 toxin gene in a zebu cow in Brazil, for the first time.(AU)
Clostridium perfringens tipo A tem sido considerado agente etiológico da síndrome do jejuno hemorrágico (SJH), que é uma doença que afeta comumente os rebanhos de gado. Embora este microrganismo seja considerado um importante enteropatógeno, a patogênese da SJH ainda não foi elucidada, e não havia sido reportada no Brasil até então. O alvo deste estudo foi descrever pela primeira vez a ocorrência da SJH causadapor C. perfringenstipo A, carreador do gene da toxina beta-2, em um zebuíno no Brasil.(AU)
Subject(s)
Animals , Cattle , Cattle Diseases , Jejunal Diseases/veterinary , Clostridium perfringens , Clostridium Infections/veterinaryABSTRACT
Toxigenic strains of Clostridium difficile may be disseminating. Here we prospectively screened patients with nosocomial diarrhoea in two hospitals in Brazil. To identify C. difficile polymerase chain reaction ribotypes 027/078 strains, we used high resolution melting and multiplex polymerase chain reaction. Among 116 screened patients, 11 were positive for C. difficile. The polymerase chain reaction ribotypes 027/078 strains were not identified in this study.