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1.
Bratisl Lek Listy ; 121(3): 182-187, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32115974

RESUMO

BACKGROUND: Clostridium (Clostridioides) difficile is the most common pathogen of nosocomial and antibiotic-related diarrhea in health-care facilities. The aim of the analysis was to show the testing algorithm and to identify hypervirulent strains (suspected RT 027). METHODS: The retrospective analysis of patient samples suspected on CDI was carried out by a two-step algorithm. Biological specimens were analysed by GDH or culture, immunoenzymatic assay on toxins A/B and selected samples also by a real-time PCR. RESULTS: In 1006 specimen suspected on CDI, 202 specimens were evaluated as positive in the two-step algorithm. Conflicting results (64 C. difficile isolates) were tested in a three-step algorithm by a real-time PCR and revealed 59 toxigenic and non RT 027 ribotypes. Statistically significant dependence among the independent variables, such as: diagnostic parameters and length of hospitalization (p = 0.175) and C. difficile (suspected RT027) ribotypes was not found. CONCLUSION: The results of PCR ribotyping showed a high prevalence of hypervirulent and toxigenic ribotypes in the studied sample. A resistance to vancomycin was found in one isolate. The PCR method contributed to the rapid laboratory diagnosis and thus treatment of high risk patients or was used as a third step in in the case of unclear results of standard diagnostic methods(Tab. 1, Fig. 4, Ref. 18). Text in PDF www.elis.sk.


Assuntos
Infecções por Clostridium , Clostridium difficile , Ribotipagem , Algoritmos , Proteínas de Bactérias/análise , Proteínas de Bactérias/genética , Infecções por Clostridium/diagnóstico , Clostridium difficile/genética , Humanos , Estudos Retrospectivos
2.
Int J Infect Dis ; 90: 111-115, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31707136

RESUMO

AIM: To obtain standardized epidemiological data for Clostridium difficile infection (CDI) in Slovakia. METHODS: Between October and December 2016, 36 hospitals in Slovakia used the European Centre for Disease Prevention and Control (ECDC) Clostridium difficile infection (CDI) surveillance protocol. RESULTS: The overall mean CDI incidence density was 2.8 (95% confidence interval 1.9-3.9) cases per 10 000 patient-days. Of 332 CDI cases, 273 (84.9%) were healthcare-associated, 45 (15.1%) were community-associated, and 14 (4.2%) were cases of recurrent CDI. A complicated course of CDI was reported in 14.8% of cases (n=51). CDI outcome data were available for 95.5% of cases (n=317). Of the 35 patients (11.1%) who died, 34 did so within 30 days after their CDI diagnosis. Of the 78 isolates obtained from 12 hospitals, 46 belonged to PCR ribotype 001 (59.0%; 11 hospitals) and 23 belonged to ribotype 176 (29.5%; six hospitals). A total of 73 isolates (93.6%) showed reduced susceptibility to moxifloxacin (ribotypes 001 and 176; p< 0.01). A reduced susceptibility to metronidazole was observed in 13 isolates that subsequently proved to be metronidazole-susceptible when, after thawing, they were retested using the agar dilution method. No reduced susceptibility to vancomycin was found. CONCLUSIONS: These results show the emergence of C. difficile ribotypes 027 and 176 with a predominance of ribotype 001 in Slovakia in 2016. Given that an almost homogeneous reduced susceptibility to moxifloxacin was detected in C. difficile isolates, this stresses the importance of reducing fluoroquinolone prescriptions in Slovak healthcare settings.


Assuntos
Infecções por Clostridium/epidemiologia , Infecções por Clostridium/microbiologia , Clostridium difficile/classificação , Adolescente , Idoso , Antibacterianos/farmacologia , Clostridium difficile/efeitos dos fármacos , Clostridium difficile/genética , Clostridium difficile/isolamento & purificação , Feminino , Humanos , Incidência , Lactente , Masculino , Moxifloxacina/farmacologia , Ribotipagem , Eslováquia/epidemiologia
3.
Emerg Microbes Infect ; 9(1): 42-52, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31873046

RESUMO

Clostridioides difficile causes healthcare-related diarrhoea in high-income countries. Highly resistant spores persist in healthcare facilities, primarily infecting patients who have recently received antimicrobials. C. difficile infection (CDI) has been studied in detail in North America and Europe; however, the epidemiology of CDI elsewhere, including the Asia-Pacific region, is largely unknown. A survey of CDI was performed in 13 Asia-Pacific countries. Epidemiological data on 600 cases were collected and molecular typing undertaken on 414 C. difficile isolates. Healthcare facility-associated CDI comprised 53.6% of cases, while community-associated CDI was 16.5%. The median age of cases was 63.0 years and 45.3% were female, 77.5% had used antibiotics in the previous 8 weeks, most frequently third-generation cephalosporins (31.7%), and 47.3% had used proton pump inhibitors. Recurrence (9.1%) and mortality (5.2%) rates were low, while complications including colitis or pseudomembranous colitis (13.8%), colectomy (0.4%), and toxic megacolon (0.2%) were uncommon. Common C. difficile strains were ribotypes 017 (16.7%), 014/020 (11.1%) and 018 (9.9%), with wide variation between countries. Binary toxin-positive strains of C. difficile were detected rarely. Overall, disease severity appeared mild, and mortality and recurrence were low. Continued education about, and surveillance of, CDI in Asia are required to reduce the burden of disease.


Assuntos
Infecções por Clostridium/epidemiologia , Infecções por Clostridium/microbiologia , Clostridium difficile/isolamento & purificação , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Ásia/epidemiologia , Austrália/epidemiologia , Infecções por Clostridium/tratamento farmacológico , Infecções por Clostridium/etiologia , Clostridium difficile/classificação , Clostridium difficile/genética , Clostridium difficile/fisiologia , Diarreia/microbiologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fatores de Risco , Adulto Jovem
4.
Lett Appl Microbiol ; 70(1): 29-35, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31631350

RESUMO

The objective of this study was to undertake a microbiological survey of foods, animal faeces and wastewater samples for Clostridium difficile, and determine the genotypes and antimicrobial susceptibilities of isolates. A total of 211 samples were tested for C. difficile using culture methods. Thirteen toxigenic C. difficile isolates were obtained; ten from wastewater samples, one each from pig and duck faeces and another from a raw meat product. Eight PCR-ribotypes (RTs) were identified, including two novel RTs (878 and 879). Single-nucleotide polymorphism analysis using WGS data for all isolates provided greater discrimination between C. difficile isolates within the same RT and multilocus sequence typing (MLST) profiles. All C. difficile isolates were found to be susceptible to the first-line human antimicrobials used to treat C. difficile infection. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to report the isolation of Clostridium difficile from animals, food and wastewater in New Zealand (NZ) and provides important data with respect to ribotypes and multilocus sequence typing profiles, whole genome sequence and antimicrobial susceptibilities. The results highlight the need for further investigations into the epidemiology of C. difficile in NZ and to elucidate the role of the environmental and food sources as transmission routes of human infection.


Assuntos
Clostridium difficile/isolamento & purificação , Fezes/microbiologia , Carne/microbiologia , Águas Residuárias/microbiologia , Animais , Antibacterianos/farmacologia , Clostridium difficile/classificação , Clostridium difficile/efeitos dos fármacos , Clostridium difficile/genética , Patos , Contaminação de Alimentos/análise , Genótipo , Humanos , Testes de Sensibilidade Microbiana , Tipagem de Sequências Multilocus , Nova Zelândia , Suínos
5.
BMC Infect Dis ; 19(1): 961, 2019 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-31711425

RESUMO

BACKGROUND: Clostridioides difficile is considered the main pathogen responsible for hospital-acquired infections. This prospective study determined the prevalence, molecular epidemiological characteristics, and risk factors for C. difficile infection (CDI) and C. difficile colonization (CDC) among patients in the intensive care unit (ICU) of a large-scale tertiary hospital in China, with the aim of providing strategies for efficient CDI and CDC prevention and control. METHODS: Stool samples were collected and anaerobically cultured for C. difficile detection. The identified isolates were examined for toxin genes and subjected to multilocus sequence typing. Patients were classified into CDI, CDC, and control groups, and their medical records were analyzed to determine the risk factors for CDI and CDC. RESULTS: Of the 800 patients included in the study, 33 (4.12%) and 25 (3.12%) were identified to have CDI and CDC, respectively. Associations with CDI were found for fever (OR = 13.993), metabolic disorder (OR = 7.972), and treatment with fluoroquinolone (OR = 42.696) or combined antibiotics (OR = 2.856). CDC patients were characterized by prolonged hospital stay (OR = 1.137), increased number of comorbidities (OR = 36.509), respiratory diseases (OR = 0.043), and treatment with vancomycin (OR = 18.168). Notably, treatment with metronidazole was found to be a protective factor in both groups (CDI: OR = 0.042; CDC: OR = 0.013). Eighteen sequence types (STs) were identified. In the CDI group, the isolated strains were predominantly toxin A and toxin B positive (A + B+) and the epidemic clone was genotype ST2. In the CDC group, the dominant strains were A + B+ and the epidemic clone was ST81. CONCLUSIONS: The prevalences of CDC and CDI in our ICU were relatively high, suggesting the importance of routine screening for acquisition of C. difficile. Future prevention and treatment strategies for CDC and CDI should consider hospital stay, enteral nutrition, underlying comorbidities, and use of combined antibiotics. Moreover, metronidazole may be a protective factor for both CDI and CDC, and could be used empirically.


Assuntos
Infecções por Clostridium/epidemiologia , Clostridium difficile/isolamento & purificação , Unidades de Terapia Intensiva , Adulto , Idoso , Idoso de 80 Anos ou mais , Antibacterianos/uso terapêutico , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , China/epidemiologia , Infecções por Clostridium/tratamento farmacológico , Infecções por Clostridium/prevenção & controle , Clostridium difficile/genética , Comorbidade , Enterotoxinas/metabolismo , Feminino , Genótipo , Humanos , Tempo de Internação , Masculino , Programas de Rastreamento , Metronidazol/uso terapêutico , Pessoa de Meia-Idade , Epidemiologia Molecular , Tipagem de Sequências Multilocus , Prevalência , Estudos Prospectivos , Fatores de Risco
6.
Vet Microbiol ; 239: 108433, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31767096

RESUMO

Overgrowth of enteric clostridia in dysbiosis in horses with colic is presumed but scarcely investigated. The objective was to provide prevalence data of Clostridium difficile and Clostridium perfringens in horses with and without gastrointestinal disease in Switzerland, and investigate microbiota differences between C. difficile shedders and non-shedders. Fecal samples were taken from healthy horses (n = 103), horses with colic (n = 98) and horses with diarrhea (n = 151). Colic horses were sampled on three days. Selective enrichment culture and molecular typing for C. difficile and C. perfringens was performed. Microbiota differences between horses with colic shedding (n = 7) and not shedding (n = 7) C. difficile were assessed using metagenomic sequencing. The cumulative prevalence (19% C. difficile; 16% C. perfringens) was higher compared to single day samples (1-10% C. difficile; 3-8% C. perfringens, all p < 0.003). Horses with colic shed significantly more C. difficile (p < 0.001) but not C. perfringens (p = 0.09) compared to healthy horses. Prevalence in horses with diarrhea was 8% for both Clostridium species. There were no significant microbiota differences between C. difficile shedders and non-shedders with regards to relative abundance on any phylogenetic level, and alpha diversity. Limited differences were seen on LEfSE analysis and in beta diversity indices. Multiple fecal samples should be taken when investigating shedding of enteric clostridia. As horses with colic shed more enteric clostridia compared to healthy horses special biosecurity protocols for horses with colic should be considered in hospitals. Differences in microbiota composition between C. difficile shedders and non-shedders were limited. Further studies on the role of dysbiosis in C. difficile are needed.


Assuntos
Infecções por Clostridium/veterinária , Clostridium difficile , Clostridium perfringens , Gastroenteropatias/veterinária , Doenças dos Cavalos/epidemiologia , Doenças dos Cavalos/microbiologia , Animais , Infecções por Clostridium/epidemiologia , Infecções por Clostridium/microbiologia , Clostridium difficile/classificação , Clostridium difficile/genética , Clostridium perfringens/classificação , Clostridium perfringens/genética , Gastroenteropatias/epidemiologia , Gastroenteropatias/microbiologia , Cavalos , Tipagem Molecular , Prevalência , Suíça/epidemiologia
7.
BMC Genomics ; 20(1): 796, 2019 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-31666016

RESUMO

BACKGROUND: Clade 5 Clostridioides difficile diverges significantly from the other clades and is therefore, attracting increasing attention due its great heterogeneity. In this study, we used third-generation sequencing techniques to sequence the complete whole genomes of three ST11 C. difficile isolates, RT078 and another two new ribotypes (RTs), obtained from three independent hospitalized elderly patients undergoing antibiotics treatment. Mobile genetic elements (MGEs), antibiotic-resistance, drug resistance genes, and virulent-related genes were analyzed and compared within these three isolates. RESULTS: Isolates 10,010 and 12,038 carried a distinct deletion in tcdA compared with isolate 21,062. Furthermore, all three isolates had identical deletions and point-mutations in tcdC, which was once thought to be a unique characteristic of RT078. Isolate 21,062 (RT078) had a unique plasmid, different numbers of transposons and genetic organization, and harboring special CRISPR spacers. All three isolates retained high-level sensitivity to 11 drugs and isolate 21,062 (RT078) carried distinct drug-resistance genes and loss of numerous flagellum-related genes. CONCLUSIONS: We concluded that capillary electrophoresis based PCR-ribotyping is important for confirming RT078. Furthermore, RT078 isolates displayed specific MGEs, indicating an independent evolutionary process. In the further study, we could testify these findings with more RT078 isolates of divergent origins.


Assuntos
Clostridium difficile/genética , Elementos de DNA Transponíveis , Evolução Molecular , Genoma Bacteriano , Clostridium difficile/isolamento & purificação , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Farmacorresistência Bacteriana/genética , Sequenciamento Completo do Genoma
8.
Emerg Microbes Infect ; 8(1): 1553-1562, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31662120

RESUMO

Molecular epidemiology of Clostridium difficile infection (CDI) has been extensively studied in North America and Europe; however, limited data on CDI are available in the Asia-Pacific region. A multicentre retrospective study was conducted in this region. C. difficile isolates were subjected to multilocus sequence typing (ST) and antimicrobial susceptibility testing. Totally, 394 isolates were collected from Hangzhou, Hong Kong, China; Busan, South Korea; Fukuoka, Japan; Singapore; Perth, Sydney, Australia; New York, the United States. C. difficile isolates included 337 toxin A-positive/B-positive/binary toxin-negative (A+B+CDT-), 48 A-B+CDT-, and nine A+B+CDT+. Distribution of dominant STs varied geographically with ST17 in Fukuoka (18.6%), Busan (56.0%), ST2 in Sydney (20.4%), Perth (25.8%). The antimicrobial resistance patterns were significantly different among the eight sites (χ2 = 325.64, p < 0.001). Five major clonal complexes correlated with unique antimicrobial resistances. Healthcare-associated (HA) CDI was mainly from older patients with more frequent antimicrobial use and higher A-B+ positive rates. Higher resistance to gatifloxacin, tetracycline, and erythromycin were observed in HA-CDI patients (χ2 = 4.76-7.89, p = 0.005-0.029). In conclusion, multiple C. difficile genotypes with varied antimicrobial resistance patterns have been circulating in the Asia-Pacific region. A-B+ isolates from older patients with prior antimicrobial use were correlated with HA-CDI.


Assuntos
Antibacterianos/farmacologia , Infecções por Clostridium/microbiologia , Clostridium difficile/efeitos dos fármacos , Farmacorresistência Bacteriana , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Ásia , Criança , Pré-Escolar , Clostridium difficile/classificação , Clostridium difficile/genética , Clostridium difficile/isolamento & purificação , Infecção Hospitalar/microbiologia , Eritromicina/farmacologia , Feminino , Gatifloxacina/farmacologia , Humanos , Lactente , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Filogenia , Tetraciclina/farmacologia , Adulto Jovem
9.
PLoS Biol ; 17(10): e3000379, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31658249

RESUMO

Recent work has revealed that Clostridioides difficile, a major cause of nosocomial diarrheal disease, exhibits phenotypic heterogeneity within a clonal population as a result of phase variation. Many C. difficile strains representing multiple ribotypes develop two colony morphotypes, termed rough and smooth, but the biological implications of this phenomenon have not been explored. Here, we examine the molecular basis and physiological relevance of the distinct colony morphotypes produced by this bacterium. We show that C. difficile reversibly differentiates into rough and smooth colony morphologies and that bacteria derived from the isolates display discrete motility behaviors. We identified an atypical phase-variable signal transduction system consisting of a histidine kinase and two response regulators, named herein colony morphology regulators RST (CmrRST), which mediates the switch in colony morphology and motility behaviors. The CmrRST-regulated surface motility is independent of flagella and type IV pili, suggesting a novel mechanism of cell migration in C. difficile. Microscopic analysis of cell and colony structure indicates that CmrRST promotes the formation of elongated bacteria arranged in bundled chains, which may contribute to bacterial migration on surfaces. In a hamster model of acute C. difficile disease, the CmrRST system is required for disease development. Furthermore, we provide evidence that CmrRST phase varies during infection, suggesting that the intestinal environment impacts the proportion of CmrRST-expressing C. difficile. Our findings indicate that C. difficile employs phase variation of the CmrRST signal transduction system to generate phenotypic heterogeneity during infection, with concomitant effects on bacterial physiology and pathogenesis.


Assuntos
Proteínas de Bactérias/genética , Clostridium difficile/metabolismo , Regulação Bacteriana da Expressão Gênica , Histidina Quinase/genética , Transdução de Sinais/genética , Animais , Proteínas de Bactérias/metabolismo , Células Clonais , Infecções por Clostridium/microbiologia , Infecções por Clostridium/patologia , Clostridium difficile/genética , Clostridium difficile/patogenicidade , Clostridium difficile/ultraestrutura , Cricetulus , Modelos Animais de Doenças , Fímbrias Bacterianas/metabolismo , Fímbrias Bacterianas/ultraestrutura , Flagelos/metabolismo , Flagelos/ultraestrutura , Histidina Quinase/metabolismo , Humanos , Movimento , Fenótipo , Ribotipagem
10.
Vet Microbiol ; 237: 108408, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31585650

RESUMO

Information on the epidemiology of C. difficile infection (CDI) in South-East Asian countries is limited, as is data on possible animal reservoirs of C. difficile in the region. We investigated the prevalence and molecular epidemiology of C. difficile in piglets and the piggery environment in Thailand and Malaysia. Piglet rectal swabs (n = 224) and piggery environmental specimens (n = 23) were collected between 2015 and 2016 from 11 farms located in Thailand and Malaysia. All specimens were tested for the presence of C. difficile with toxigenic culture. PCR assays were performed on isolates to determine the ribotype (RT), and the presence of toxin genes. Whole genome sequencing was used on a subset of isolates to determine the evolutionary relatedness of RT038 (the most prevalent RT identified) common to pigs and humans from Thailand and Indonesia. C. difficile was recovered from 35% (58/165) and 92% (54/59) of the piglets, and 89% (8/9) and 93% (13/14) of the environmental specimens from Thailand and Malaysia, respectively. All strains from Thailand, and 30 strains from Malaysia (23 piglet and 7 environmental isolates) were non-toxigenic. To our knowledge, this is the first and only report with a complete lack of toxigenic C. difficile among piglets, a feature which could have a protective effect on the host. The most common strain belonged to RT038 (ST48), accounting for 88% (51/58) of piglet and 78% (7/9) of environmental isolates from Thailand, and all 30 isolates tested from Malaysia. Piglet RT038 isolates from Thailand and Malaysia differed by only 18 core-genome single nucleotide variants (cgSNVs) and both were, on average, 30 cgSNVs different from the human strains from Thailand and Indonesia, indicating a common ancestor in the last two decades.


Assuntos
Infecções por Clostridium/veterinária , Clostridium difficile/genética , Doenças dos Suínos/microbiologia , Animais , Infecções por Clostridium/epidemiologia , Infecções por Clostridium/microbiologia , Microbiologia Ambiental , Variação Genética , Abrigo para Animais , Malásia/epidemiologia , Epidemiologia Molecular , Prevalência , Suínos , Tailândia/epidemiologia , Sequenciamento Completo do Genoma
12.
Biomed Res Int ; 2019: 4278598, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31380423

RESUMO

Rhodococcus equi is responsible for infections in multiple-host animals. In humans, the prevalence of rhodococcus has increased worldwide and represents an emergent risk. R. equi is a soil-borne opportunistic bacterium isolated from feces of a wide variety of domestic species, except cats; thus, there is no known potential risk of its transmission from humans. Here, the mono- and cooccurrence of Rhodococcus equi and other bacteria and selected virulence markers were investigated in feces of nondiarrheic cats from urban (n=100) and rural (n=100) areas. Seven (7/200=3.5%) R. equi isolates were recovered in ceftazidime, novobiocin, and cycloheximide (CAZ-NB) selective media, exclusively of cats from three distinct farms (p=0.01), and these cats had a history of contact with horses and their environment (p=0.0002). None of the R. equi isolates harbored hosted-adapted plasmid types associated with virulence (pVAPA, pVAPB, and pVAPN). One hundred seventy-five E. coli isolates were identified, and 23 atypical enteropathogenic E. coli (aEPEC), 1 STEC (Shiga-toxin producing E. coli), and 1 EAEC (enteroaggregative E. coli) were detected. Eighty-six C. perfringens type A isolates were identified, and beta-2 and enterotoxin were detected in 21 and 1 isolates, respectively. Five C. difficile isolates were identified, one of which was toxigenic and ribotype 106. The main cooccurring isolates in cats from urban areas were E. coli and C. perfringens A (26/100=26%), E. coli and C. perfringens type A cpb2+ (8/100=8%), and aEPEC (eae+/escN+) and C. perfringens type A (5/100=5%). In cats from farms, the main cooccurring isolates were E. coli and C. perfringens type A (21/100=21%), E. coli and C. perfringens type A cpb 2 + 8/100=8%), and E. coli and R. equi (4/100=4%). We identified, for the first time, R. equi in nondiarrheic cats, a finding that represents a public health issue because rhodococcus has been reported in both immunosuppressed and immunocompetent humans, particularly people living with HIV/AIDS.


Assuntos
Gatos/microbiologia , Fezes/microbiologia , Rhodococcus equi/isolamento & purificação , Microbiologia do Solo , Animais , Clostridium difficile/genética , Clostridium difficile/isolamento & purificação , Escherichia coli/genética , Escherichia coli/isolamento & purificação , Cavalos/microbiologia , Humanos , Rhodococcus equi/genética , Rhodococcus equi/patogenicidade
13.
Nat Genet ; 51(9): 1315-1320, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31406348

RESUMO

Bacterial speciation is a fundamental evolutionary process characterized by diverging genotypic and phenotypic properties. However, the selective forces that affect genetic adaptations and how they relate to the biological changes that underpin the formation of a new bacterial species remain poorly understood. Here, we show that the spore-forming, healthcare-associated enteropathogen Clostridium difficile is actively undergoing speciation. Through large-scale genomic analysis of 906 strains, we demonstrate that the ongoing speciation process is linked to positive selection on core genes in the newly forming species that are involved in sporulation and the metabolism of simple dietary sugars. Functional validation shows that the new C. difficile produces spores that are more resistant and have increased sporulation and host colonization capacity when glucose or fructose is available for metabolism. Thus, we report the formation of an emerging C. difficile species, selected for metabolizing simple dietary sugars and producing high levels of resistant spores, that is adapted for healthcare-mediated transmission.


Assuntos
Aclimatação/genética , Infecções por Clostridium/transmissão , Clostridium difficile/genética , Especiação Genética , Esporos Bacterianos/crescimento & desenvolvimento , Açúcares/metabolismo , Virulência/genética , Antibacterianos/farmacologia , Infecções por Clostridium/metabolismo , Infecções por Clostridium/microbiologia , Clostridium difficile/isolamento & purificação , Genoma Bacteriano , Genômica , Humanos , Esporos Bacterianos/efeitos dos fármacos , Esporos Bacterianos/genética , Esporos Bacterianos/metabolismo
14.
mSphere ; 4(4)2019 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-31462412

RESUMO

There has been no prior application of matched metagenomics and metatranscriptomics in Clostridioides difficile infection (CDI) evaluating the role of fungi in CDI or identifying community functions that contribute to the development of this disease. We collected diarrheal stools from 49 inpatients (18 of whom tested positive for CDI) under stringent inclusion criteria. We utilized a tiered sequencing approach to identify enriched bacterial and fungal taxa, using 16S and internal transcribed spacer (ITS) rRNA gene amplicon sequencing, with matched metagenomics and metatranscriptomics performed on a subset of the population. Distinct bacterial and fungal compositions distinguished CDI-positive and -negative patients, with the greatest differentiation between the cohorts observed based on bacterial metatranscriptomics. Bipartite network analyses demonstrated that Aspergillus and Penicillium taxa shared a strong positive relationship in CDI patients and together formed negative cooccurring relationships with several bacterial taxa, including the Oscillospira, Comamonadaceae, Microbacteriaceae, and Cytophagaceae Metatranscriptomics revealed enriched pathways in CDI patients associated with biofilm production primarily driven by Escherichia coli and Pseudomonas, quorum-sensing proteins, and two-component systems related to functions such as osmotic regulation, linoleic acid metabolism, and flagellar assembly. Differential expression of functional pathways unveiled a mechanism by which the causal dysbiosis of CDI may self-perpetuate, potentially contributing to treatment failures. We propose that CDI has a distinct fungus-associated bacteriome, and this first description of metatranscriptomics in human subjects with CDI demonstrates that inflammation, osmotic changes, and biofilm production are key elements of CDI pathophysiology.IMPORTANCE Our data suggest a potential role for fungi in the most common nosocomial bacterial infection in the United States, introducing the concept of a transkingdom interaction between bacteria and fungi in this disease. We also provide the first direct measure of microbial community function in Clostridioides difficile infection using patient-derived tissue samples, revealing antibiotic-independent mechanisms by which C. difficile infection may resist a return to a healthy gut microbiome.


Assuntos
Infecções por Clostridium/microbiologia , Clostridium difficile/genética , Fungos/genética , Microbioma Gastrointestinal , Metagenômica , Transcriptoma , Adulto , Idoso , Idoso de 80 Anos ou mais , Biofilmes , Diarreia/microbiologia , Fezes/microbiologia , Humanos , Redes e Vias Metabólicas , Pessoa de Meia-Idade , Análise de Sequência de DNA
15.
Zoonoses Public Health ; 66(7): 861-870, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31389666

RESUMO

Swine are known reservoirs for Clostridioides difficile, formerly known as Clostridium difficile, and transmission from swine to human farm workers is strongly suggested by previous studies. This cross-sectional study evaluated the potential role of farm environmental surfaces, including those in worker breakrooms and swine housing areas, in the possible transmission of C. difficile from swine to farm workers. Environmental surfaces and piglet faeces at 13 Ohio swine farms were sampled in 2015. Typical culturing techniques were performed to isolate C. difficile from samples, and amplification of toxin genes (tcdA, tcdB and cdtB) and PCR-ribotyping were used to genetically characterize recovered isolates. In addition, sequencing of toxin regulatory gene, tcdC, was done to identify the length of identified deletions in some isolates. A survey collected farm-level management risk factor information. Clostridioides difficile was recovered from all farms, with 42% (188/445) of samples testing positive for C. difficile. Samples collected from all on-farm locations recovered C. difficile, including farrowing rooms (60%, 107/178), breakrooms (50%, 69/138) and nursery rooms (9%, 12/129). Three ribotypes recovered from both swine and human environments (078, 412 and 005) have been previously implicated in human disease. Samples taken from farrowing rooms and breakrooms were found to have greater odds of C. difficile recovery than those taken from nursery rooms (OR = 40.5, OR = 35.6, p < .001 respectively). Farms that weaned ≥23,500 pigs per year had lower odds of C. difficile recovery as compared to farms that weaned fewer pigs (OR = 0.4, p = .01) and weekly or more frequent cleaning of breakroom counters was associated with higher odds of C. difficile recovery (OR = 11.7, p < .001). This study provides important insights into the presence and characterization of C. difficile found in human environments on swine farms and highlights how these areas may be involved in transmission of C. difficile to swine farm workers and throughout the facility.


Assuntos
Infecções por Clostridium/veterinária , Fazendas , Doenças dos Suínos/microbiologia , Animais , Infecções por Clostridium/microbiologia , Clostridium difficile/genética , Microbiologia Ambiental , Fezes , Abrigo para Animais , Humanos , Ohio/epidemiologia , Fatores de Risco , Suínos , Zoonoses
16.
Klin Mikrobiol Infekc Lek ; 25(1): 12-15, 2019 Mar.
Artigo em Tcheco | MEDLINE | ID: mdl-31266088

RESUMO

OBJECTIVE: Clostridioides difficile (formerly Clostridium difficile) is one of the main pathogens causing nosocomial infections today. It colonizes the intestines of patients receiving antibiotic therapy, causing unpleasant or even life-threatening conditions (diarrhea, toxic megacolon). Rapid and correct detection of strain toxigenicity is essential for treatment and isolation of patients. Simplexa C. difficile Direct Kit is a real-time PCR kit detecting the tcdB gene of C. difficile. The kit does not require DNA isolation; stool eluates are directly used for the reaction. The study aimed to verify the analytical properties of the kit by its comparison with culture and in-house multiplex PCR methods. MATERIAL AND METHODS: A total of 164 stool samples were prospectively tested using two immunoenzymatic kits (C. diff Quik Chek Complete and LIAISON C. difficile GDH, Toxins AandB). In 39 samples, the results were discrepant or unclear (GDH+TOX-). These samples were tested using in-house multiplex PCR and the Simplexa kit. RESULTS: The Simplexa kit had 94.7% sensitivity, 100% specificity, 100% positive predictive value and 95.2% negative predictive value. These parameters were calculated from the numbers of true-/false-positive and true-/false-negative results. True results were determined based on the consensus of culture and in-house multiplex PCR results. Another outcome of the study was comparison of the Quik Chek and LIAISON kits. CONCLUSION: The analytical properties of the Simplexa kit were tested on 39 samples. These samples were selected for their unclear (GDH+TOX-) or discrepant results yielded by immunoenzymatic methods. Compared with culture and subsequent in-house PCR detection of the tcdB gene, the Simplexa kit showed properties declared by the manufacturer. An important advantage of the kit was the absence inhibitions when stool eluates were directly used for PCR reactions.


Assuntos
Proteínas de Bactérias , Toxinas Bacterianas , Infecções por Clostridium , Clostridium difficile , Kit de Reagentes para Diagnóstico , Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Infecções por Clostridium/diagnóstico , Clostridium difficile/genética , Fezes/microbiologia , Humanos , Reação em Cadeia da Polimerase/normas , Kit de Reagentes para Diagnóstico/normas , Sensibilidade e Especificidade
17.
Eur J Clin Microbiol Infect Dis ; 38(10): 1953-1959, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31327067

RESUMO

Clostridioides difficile has become the leading nosocomial Gram-positive pathogen in the developed countries. In Lithuania, the national surveillance program for C. difficile started in 2017. Enzyme immunoassay, the real-time PCR system, and culture are used for laboratory confirmation of C. difficile infection in Lithuanian clinical laboratories. No reference laboratory for C. difficile is present in Lithuania. Fifty-eight isolates of C. difficile were collected in 2016 and 2017 in two hospitals using real-time PCR and culture methods. Agarose gel-based PCR ribotyping, multilocus variable number tandem repeats analysis (MLVA), and multilocus sequence typing (MLST) were used for the genotypic characterization of 28 isolates. PCR ribotyping and MLST showed that 78.6% of the tested toxigenic isolates belong to the ribotype RT027/ST1. Using MLVA, 95.5% of RT027 isolates were genetically related. MLVA revealed three clonal complexes in RT027. Six non-RT027 isolates showed four different electrophoretic profiles in PCR ribotyping and were assigned to the MLST sequence types ST2, ST13, ST54, and ST63. The highest discriminatory power showed the genotyping by MLVA. In total, 20 MLVA profiles were identified. This genotyping technique allowed to identify four groups of RT027/ST1 isolates that were indistinguishable by PCR ribotyping and MLST. Our study is the first genotypic characterization of C. difficile isolates in Lithuania. We observed a high prevalence of presumptive RT027 that suggests unfavorable epidemiological situation in Lithuania. Our results stress for implementation of genotyping of C. difficile isolates in Lithuanian surveillance.


Assuntos
Infecções por Clostridium/epidemiologia , Infecções por Clostridium/microbiologia , Clostridium difficile/classificação , Clostridium difficile/genética , Genótipo , Ribotipagem , Técnicas Bacteriológicas , Clostridium difficile/isolamento & purificação , Infecção Hospitalar/epidemiologia , Infecção Hospitalar/microbiologia , Hospitais , Humanos , Lituânia/epidemiologia , Repetições Minissatélites , Técnicas de Diagnóstico Molecular , Epidemiologia Molecular , Tipagem de Sequências Multilocus , Projetos Piloto , Prevalência
18.
Transpl Infect Dis ; 21(5): e13149, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31332916

RESUMO

BACKGROUND: Clostridioides (Clostridium) difficile infection (CDI) in pediatric solid organ transplant (SOT) recipients is a growing problem, though CDI risk factors in this population are poorly understood. Our objective was to characterize CDI risk factors in pediatric SOT recipients. METHODS: This retrospective case-control study, performed at a single freestanding academic children's hospital, included all SOT recipients age 1-22 years who were tested for C. difficile by toxin B gene PCR between August 2009 and August 2017. CDI risk factors were assessed by comparing PCR-positive and PCR-negative cases by generalized linear mixed models. RESULTS: Between August 2009 and August 2017, 409 SOTs were performed of which 138 (33.7%), 134 (32.8%), 131 (32.0%), and 6 (1.5%) were kidney, liver, heart, and small intestine transplants, respectively. Of 205 SOT recipients were tested for CDI, with 723 C. difficile PCR tests performed among these patients. 68/205 (33%) patients developed CDI at least once during the study period. Median (interquartile range) time to diagnosis of first CDI following SOT was 8.9 (1.2, 19.6) months. CDI was independently associated with calcineurin inhibitor use at time of C. difficile testing (odds ratio [OR] 2.38, 95% confidence interval [CI] 1.08, 5.24, P = 0.03) and systemic antibiotic exposure within 30 days of C. difficile testing (OR 1.74, 95% CI 1.08, 2.79, P = 0.02). CONCLUSIONS: CDI is a common, relatively late post-transplant complication and independently associated with calcineurin inhibitor and systemic antibiotic exposure. The potential impact of specific immunosuppressive drug and antibiotic selection on CDI risk reduction requires further investigation.


Assuntos
Infecções por Clostridium/etiologia , Transplante de Órgãos/efeitos adversos , Transplantados , Adolescente , Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Estudos de Casos e Controles , Criança , Pré-Escolar , Clostridium difficile/genética , Registros Eletrônicos de Saúde , Feminino , Humanos , Lactente , Masculino , Razão de Chances , Estudos Retrospectivos , Fatores de Risco , Adulto Jovem
19.
Mol Biol Evol ; 36(12): 2714-2736, 2019 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-31350897

RESUMO

Bacteria of the Firmicutes phylum are able to enter a developmental pathway that culminates with the formation of highly resistant, dormant endospores. Endospores allow environmental persistence, dissemination and for pathogens, are also infection vehicles. In both the model Bacillus subtilis, an aerobic organism, and in the intestinal pathogen Clostridioides difficile, an obligate anaerobe, sporulation mobilizes hundreds of genes. Their expression is coordinated between the forespore and the mother cell, the two cells that participate in the process, and is kept in close register with the course of morphogenesis. The evolutionary mechanisms by which sporulation emerged and evolved in these two species, and more broadly across Firmicutes, remain largely unknown. Here, we trace the origin and evolution of sporulation using the genes known to be involved in the process in B. subtilis and C. difficile, and estimating their gain-loss dynamics in a comprehensive bacterial macroevolutionary framework. We show that sporulation evolution was driven by two major gene gain events, the first at the base of the Firmicutes and the second at the base of the B. subtilis group and within the Peptostreptococcaceae family, which includes C. difficile. We also show that early and late sporulation regulons have been coevolving and that sporulation genes entail greater innovation in B. subtilis with many Bacilli lineage-restricted genes. In contrast, C. difficile more often recruits new sporulation genes by horizontal gene transfer, which reflects both its highly mobile genome, the complexity of the gut microbiota, and an adjustment of sporulation to the gut ecosystem.


Assuntos
Bacillus subtilis/genética , Evolução Biológica , Clostridium difficile/genética , Esporos Bacterianos , Sequência de Aminoácidos , Genes Bacterianos
20.
Anaerobe ; 58: 103-109, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31323291

RESUMO

The production of TcdA, TcdB and CDT in Clostridium difficile PCR ribotype 027, is regulated by the two-component system response regulator CdtR. Despite this, little is known about the signal transduction pathway leading to the activation of CdtR. In this study, we generated R20291ΔPalocΔcdtR model strains expressing CdtR phospho-variants in which our predicted phospho-accepting Asp, Asp61 was mutated for Ala or Glu. The constructs were assessed for their ability to restore CDT production. Dephospho-CdtR-Asp61Ala was completely non-functional and mirrored the cdtR-deletion mutant, whilst phospho-CdtR-Asp61Glu was functional, possessing 38-52% of wild-type activity. Taken together, these data suggest that CdtR is activated by phosphorylation of Asp61. The same principles were applied to assess the function of PCR ribotype 078-derived CdtR, which was shown to be non-functional owing to polymorphisms present within its coding gene. Conversely, polymorphisms present within its promoter region, provide significantly enhanced promoter activity compared with its PCR ribotype 027 counterpart. To ensure our data were representative for each ribotype, we determined that the cdtR nucleotide sequence was conserved in a small library of eight PCR ribotype 027 clinical isolates and nineteen PCR ribotype 078 isolates from clinical and animal origin.


Assuntos
Toxinas Bacterianas/biossíntese , Clostridium difficile/metabolismo , Regulação Bacteriana da Expressão Gênica , Processamento de Proteína Pós-Traducional , Fatores de Transcrição/metabolismo , Infecções por Clostridium , Clostridium difficile/genética , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Fosforilação , Deleção de Sequência , Transdução de Sinais , Fatores de Transcrição/genética
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