Abstract Background C. difficile has been increasingly reported as a cause of gastrointestinal disease in children, ranging from mild self-limiting diarrhea to severe conditions such as pseudomembranous colitis and toxic megacolon. Only two pediatric research groups reported the presence of C. difficile infection in Brazilian children, but no previous research has examined C. difficile infection among children in northeastern Brazil. This prospective cross-sectional study investigated the molecular epidemiology and antimicrobial resistance of C. difficile strains isolated from children and adolescents with diarrhea referred to a tertiary pediatric hospital in Brazil while exploring the associated risk factors. Results Toxin positivity or C. difficile isolation was found in 30.4 % (17/56) samples. C. difficile was isolated from 35 % (6/17) samples. Four toxigenic strains were identified (tpi+, tcdA+, tcdB+, cdtB-, without tcdC deletions) belonging to PCR ribotypes and PFGE-pulsotypes: 046 (new pulsotype 1174), 106 (NAP11), 002 (new pulsotype 1274), 012 (new pulsotype NML-1235). Two of the six isolates belonging to ribotypes 143 and 133 were non-toxigenic. All toxigenic strains were sensitive to metronidazole and vancomycin. Regarding the clinical manifestation, diarrhea lasted an average of 11 days, ranging from 3 to 50 days and was often associated with mucus and/or blood. All six patients from whom the C. difficile was isolated had a chronic disease diagnosis, with these comorbidities as the main risk factors. Conclusion Our study enhances our understanding of the present epidemiological landscape of C. difficile-associated diarrhea (CDI) among children in northeastern Brazil, reveling a substantial CDI frequency of 30.4 %, with toxigenic strains detected in 76.4 % of cases, highlighting a higher prevalence compared to earlier Brazilian studies. In the globalized world, an understanding of disease-generating strains, the associated risk factors, clinical manifestation, and antimicrobial sensitivity has fundamental epidemiological importance and draws attention to preventive measures, allowing for more decisive action. C selflimiting self limiting megacolon crosssectional cross sectional factors 304 30 4 30. 17/56 1756 17 56 (17/56 samples 6/17 617 6 (6/17 tpi+, tpi (tpi+ tcdA tcdA+ tcdB tcdB+ cdtB, cdtB , cdtB- deletions PFGEpulsotypes PFGE pulsotypes PFGE-pulsotypes 04 new 1174, 1174 1174) 10 NAP11, NAP11 NAP (NAP11) 00 1274, 1274 1274) 01 NML1235. NML1235 NML 1235 . NML-1235) 14 13 nontoxigenic. nontoxigenic non toxigenic. non-toxigenic vancomycin manifestation 1 5 andor blood diagnosis difficileassociated (CDI 764 76 76. cases studies world diseasegenerating generating measures action 17/5 175 (17/5 6/1 61 (6/1 tpi+ (tpi 0 117 NAP1 (NAP11 127 NML123 123 NML-1235 7 17/ (17/ 6/ (6/ (NAP1 12 NML12 NML-123 (17 (6 (NAP NML1 NML-12 (1 ( NML-1 NML-

BACKGROUND Clostridioides difficile is the most common cause of nosocomial diarrhea associated with antibiotic use. The disease’s symptoms are caused by enterotoxins, but other surface adhesion factors also play a role in the pathogenesis. These adhesins will bind to components of extracellular matrix. OBJECTIVE There is a lack of knowledge on MSCRAMM, this work set-out to determine the adhesive properties of several C. difficile ribotypes (027, 133, 135, 014, 012) towards laminin-1 (LMN-1). METHODS A binding experiment revealed that different ribotypes have distinct adhesion capabilities. To identify this adhesin, an affinity chromatography column containing LMN-1 was prepared and total protein extracts were analysed using mass spectrometry. FINDINGS Strains from ribotypes 012 and 027 had the best adhesion when incubated with glucose supplementations (0.2%, 0.5%, and 1%), while RT135 had a poor adherence. The criteria were not met by RT014 and RT133. In the absence of glucose, there was no adhesion for any ribotype, implying that glucose is required and plays a significant role in adhesion. MAIN CONCLUSIONS These findings show that in the presence of glucose, each C. difficile ribotype interacts differently with LMN-1, and the adhesin responsible for recognition could be SlpA protein.

Resumo Ehrlichia canis é o principal agente etiológico da erliquiose monocítica canina (EMC), uma doença infecciosa canina globalmente dispersa. No Brasil, a EMC é considerada endêmica, e a infecção pode atingir 65% em cães em alguns estados. O diagnóstico de erliquiose é importante para fins de tratamento e epidemiológicos. A proteína TRP36 de E. canis leva a uma resposta humoral com produção de anticorpos em fase aguda, encontrada durante o curso da doença. O objetivo deste estudo foi obter a proteína TRP36 recombinante da amostra São Paulo de E. canis e avaliar seu potencial como ferramenta para o diagnóstico sorológico da CME. O isolado de E. canis São Paulo foi cultivado em células da linhagem DH82 e o DNA genômico foi obtido. O fragmento de DNA bacteriano que codifica toda a ORF de TRP36 foi clonado no vetor pBAD / Thio-TOPO e transformado em células competentes Escherichia coli DH10B, com o plasmídeo portador de trp36 para expressão de proteínas. Para avaliar a antigenicidade da proteína, 16 amostras de soro canino foram previamente analisadas (por PCR e teste sorológico comercial SNAP®4Dx®). Os resultados estavam de acordo com o teste SNAP®4Dx®. Os experimentos que utilizam essa proteína recombinante como antígeno, visando ao desenvolvimento de um teste sorológico baseado no ELISA, são o próximo passo para produzir um teste de diagnóstico confiável, acessível e útil para o diagnóstico da EMC no Brasil.

Abstract Ehrlichia canis is the main etiological agent of canine monocytic ehrlichiosis (CME), a globally canine infectious disease. In Brazil, CME is considered to be endemic, and its prevalence can reach 65% in some states. The diagnosis of ehrlichiosis is important for treatment and epidemiological purposes. The E. canis TRP36 (Tandem Repeat Protein) protein elicits the earliest acute-phase antibody response observed during the course of the disease. This study aimed to generate the recombinant TRP36 protein from E. canis São Paulo strain and to evaluate its potential as a tool for the serologic diagnosis of CME. The E. canis São Paulo isolate was cultivated in DH82 lineage cells, and its genomic DNA was obtained. The bacterial DNA fragment encoding the entire ORF of TRP36 was cloned into the pBAD/Thio-TOPO vector and transformed into Escherichia coli DH10B competent cells with the trp36-bearing plasmid for protein expression. To evaluate the protein antigenicity, 16 canine serum samples were previously tested (by PCR and the commercial SNAP®4Dx® serological test). The results were in accordance with the SNAP®4Dx® test. Experiments using this recombinant protein as an antigen, targeting the development of a serologic test based on ELISA methodology, are the next step to produce a reliable, affordable and useful diagnostic tool for CME in Brazil.

BACKGROUND Members of the Bacteroides fragilis group are the most important components of the normal human gut microbiome, but are also major opportunistic pathogens that are responsible for significant mortality, especially in the case of bacteraemia and other severe infections, such as intra-abdominal abscesses. Up to now, several virulence factors have been described that might explain the involvement of B. fragilis in these infections. The secretion of extracellular membrane vesicles (EMVs) has been proposed to play a role in pathogenesis and symbiosis in gram-negative bacteria, by releasing soluble proteins and other molecules. In B. fragilis, these vesicles are known to have haemagglutination and sialidosis activities, and also contain a capsular polysaccharide (PSA), although their involvement in virulence is still not clear. OBJECTIVE The aim of this study was to identify proteins in the EMV of the 638R B. fragilis strain by mass spectrometry, and also to assess for the presence of Bfp60, a surface plasminogen (Plg) activator, previously shown in B. fragilis to be responsible for the conversion of inactive Plg to active plasmin, which can also bind to laminin-1. METHODS B. fragilis was cultured in a minimum defined media and EMVs were obtained by differential centrifugation, ultracentrifugation, and filtration. The purified EMVs were observed by both transmission electron microscopy (TEM) and immunoelectron microscopy (IM). To identify EMV constituent proteins, EMVs were separated by 1D SDS-PAGE and proteomic analysis of proteins sized 35 kDa to approximately 65 kDa was performed using mass spectrometry (MALDI-TOF MS). FINDINGS TEM micrographs proved the presence of spherical vesicles and IM confirmed the presence of Bfp60 protein on their surface. Mass spectrometry identified 23 proteins with high confidence. One of the proteins from the B. fragilis EMVs was identified as an enolase P46 with a possible lyase activity. MAIN CONCLUSIONS Although the Bfp60 protein was not detected by proteomics, α-enolase P46 was found to be present in the EMVs of B. fragilis. The P46 protein has been previously described to be present in the outer membrane of B. fragilis as an iron-regulated protein.

O objetivo deste estudo foi caracterizar as cepas de Ehrlichia canis em cães naturalmente infectados no Rio de Janeiro, Brasil. Além disso, os achados clínicos e hematológicos observados nos cães foram relatados. O gene 16S rRNA foi utilizado como alvo da PCR para fins diagnósticos, e os genes TRP19 e TRP36 para avaliar a diversidade genética. Quinze amostras foram positivas para E. canis. PCR para o gene TRP19 produziu 11 amplicons (11/15) que foram clonados no pGEM-T easy vector para sequenciamento. A comparação das sequências completas do gene TRP19 com outras sequências depositadas no GenBank revelou uma alta identidade. Duas amostras (56C e 70C) após o ensaio da PCR, tendo como alvo o gene TRP36, geraram sequências, e a análise filogenética mostrou que a cepa 56C foi agrupada com a cepa Cuiabá 16, que é uma cepa híbrida, formada pelo genogrupo Brasileiro e o genogrupo US; e a cepa 70C agrupou com as outras cepas do genogrupo US, sugerindo a existência de pelo menos dois genogrupos de E. canis no Rio de Janeiro (US e Brasileiro). Esses animais apresentaram manifestações clínicas e hematológicas distintas, e diferentes genótipos podem expressar novos fenótipos, resultando em diferentes formas de apresentação da doença e fazendo com que o diagnóstico seja mais complexo.

The aim of this study was to characterize Ehrlichia canis strains from naturally infected dogs in Rio de Janeiro, Brazil. In addition, all the clinical and hematological findings observed in these dogs were reported. PCR targeting the 16S rRNA gene was used for diagnostic purposes, and the TRP19 and TRP36 genes were sequenced to evaluate the genetic diversity. Fifteen samples were positive for E. canis. The polymerase chain reaction for the TRP19 gene resulted in 11 amplicons (11/15), which were cloned into the pGEM-T easy vector for sequencing. The complete sequence of TRP19 gene was compared to those in the GenBank, revealing high identicalness. Phylogenetic analysis on the TRP36 gene sequences demonstrated two distinct strains from two dogs, named 56C and 70C. The 56C strain was grouped with the strain Cuiaba 16, which is a hybrid strain formed by Brazilian and US genogroups; and the 70C strain was grouped with other strains of the US genogroup, thus suggesting that there are at least two genogroups of E. canis in Rio de Janeiro (US and Brazilian). Those animals, in which the 70C and 56C strains were isolated, showed distinct clinical and hematological manifestations of 1the disease. The appearance of different genotypes may express new phenotypes, thus resulting in different forms of presentation of the disease and making its diagnosis more complex.

The Bacteroides fragilis ATCC strain was grown in a synthetic media with contrasting redox potential (Eh) levels [reduced (-60 mV) or oxidised (+100mV)] and their adhesion capacity to extracellular matrix components was evaluated. The strain was capable of adhering to laminin, fibronectin, fibronectin + heparan sulphate and heparan sulphate. A stronger adherence to laminin after growing the strain under oxidising conditions was verified. Electron microscopy using ruthenium red showed a heterogeneous population under this condition. Dot-blotting analyses confirmed stronger laminin recognition by outer membrane proteins of cells cultured at a higher Eh. Using a laminin affinity column, several putative laminin binding proteins obtained from the cultures kept under oxidising (60 kDa, 36 kDa, 25 kDa and 15 kDa) and reducing (60 kDa) conditions could be detected. Our results show that the expression of B. fragilis surface components that recognise laminin are influenced by Eh variations.