Molecular epidemiology and antimicrobial resistance in Clostridioides difficile strains isolated from children and adolescents in a tertiary referral pediatric hospital in Fortaleza, Brazil.

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.

Biofilm formation by Staphylococcus pseudintermedius on titanium implants.

Osteomyelitis often involves Staphylococcus spp. as the isolated genus in domestic animal cases. Implant-related infections, frequently associated with biofilm-forming microorganisms like staphylococci species, necessitate careful material selection. This study assessed biofilm formation by Staphylococcus pseudintermedius on titanium nuts used in veterinary orthopaedic surgery. Biofilm quantification employed safranin staining and spectrophotometric measurement, while bacterial counts were determined in colony-forming units (CFU). Scanning Electron Microscopy (SEM) evaluated the biofilm morphology on the surface of titanium nuts. All samples had CFU counts. Absorbance values that evidence biofilm formation were observed in seven of the eight samples tested. SEM images revealed robust bacterial colonization, and significant extracellular polymeric substance production, and the negative control displayed surface irregularities on the nut. Whole genome sequencing revealed accessory Gene Regulator (agr) type III in six samples, agr IV and agr II in two each. Genes encoding hlb, luk-S, luk-F, siet, se_int, and the icaADCB operon were identified in all sequenced samples. Other exfoliative toxins were absent. Biofilm formation by S. pseudintermedius was detected in all samples, indicating the susceptibility of orthopaedic titanium alloys to adhesion and biofilm formation by veterinary species. The biofilm formation capacity raises concerns about potential post-surgical complications and associated costs.

Prevalence of Clostridioides difficile in dogs (Canis familiaris) with gastrointestinal disorders in Rio de Janeiro.

Clostridioides difficile infections (CDI) have a high morbidity and mortality rate and have always been considered a nosocomial disease. Nonetheless, the number of cases of community-acquired CDI is increasing, and new evidence suggests additional C. difficile reservoirs exist. Pathogenic C. difficile strains have been found in livestock, domestic animals, and meat, so a zoonotic transmission has been proposed. OBJECTIVE: The goal of this study was to isolate C. difficile strains in dogs at a veterinary clinic in Rio de Janeiro, Brazil, and characterize clinical and pathological findings associated with lower gastrointestinal tract disorders. METHODS: Fifty stool samples and biopsy fragments from dogs were obtained and cultured in the CDBA selective medium. All suggestive C. difficile colonies were confirmed by MALDI-TOF MS and PCR (tpi gene). Vancomycin, metronidazole, moxifloxacin, erythromycin, and rifampicin were tested for antibiotic susceptibility. Biofilm, motility assays, and a PCR for the toxins (tcdA, tcdB, and cdtB), as well as ribotyping, were also performed. RESULTS: Blood samples and colonic biopsy fragments were examined in C. difficile positive dogs. Ten animals (20%) tested positive for C. difficile by using stool samples, but not from biopsy fragments. Most C. difficile strains were toxigenic: six were A+B+ belonging to RT106; two were A+B+ belonging to RT014/020; and two were A-B- belonging to RT010. All strains were biofilm producers. In the motility test, 40% of strains were as motile as the positive control, CD630 (RT012). In the disc diffusion test, two strains (RT010) were resistant to erythromycin and metronidazole; and another to metronidazole (RT014/020). In terms of C. difficile clinicopathological correlations, no statistically significant morphological changes, such as pseudomembranous and "volcano" lesions, were observed. Regarding hematological data, dogs positive for C. difficile had leucopenia (p = 0.02) and lymphopenia (p = 0.03). There was a significant correlation between senility and the presence of C. difficile in the dogs studied (p = 0,02). CONCLUSIONS: Although C. difficile has not been linked to canine diarrheal disorders, it appears to be more common in dogs with intestinal dysfunctions. The isolation of ribotypes frequently involved in human CDI outbreaks around the world supports the theory of C. difficile zoonotic transmission.

Colonization of intervertebral discs by Cutibacterium acnes in patients with low back pain: Protocol for an analytical study with microbiological, phenotypic, genotypic, and multiomic techniques.

Lumbar disc degeneration (LDD) and low back pain (LBP) are two conditions that are closely related. Several studies have shown Cutibacterium acnes colonization of degenerated discs, but whether and how these finding correlates with LBP is unknown. A prospective study was planned to identify molecules present in lumbar intervertebral discs (LLIVD) colonized by C. acnes in patients with LDD and LBP and correlate them with their clinical, radiological, and demographic profiles. The clinical manifestations, risk factors, and demographic characteristics of participants undergoing surgical microdiscectomy will be tracked. Samples will be isolated and pathogens found in LLIVD will be characterized phenotypically and genotypically. Whole genome sequencing (WGS) of isolated species will be used to phylotype and detect genes associated with virulence, resistance, and oxidative stress. Multiomic analyses of LLIVD colonized and non-colonized will be carried out to explain not only the pathogen's role in LDD, but also its involvement in the pathophysiology of LBP. This study was approved by the Institutional Review Board (CAAE 50077521.0.0000.5258). All patients who agree to participate in the study will sign an informed consent form. Regardless of the study's findings, the results will be published in a peer-reviewed medical journal. Trials registration number NCT05090553; pre-results.

Binding of the extracellular matrix laminin-1 to Clostridioides difficile strains.

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.

Comparative biofilm-forming ability between Clostridioides difficile strains isolated in Latin America and the epidemic NAP1/027 strain.

Introduction: One of the challenges in treating Clostridioides difficile infection (CDI) is that the bacterium forms biofilms, a critical virulence mechanism known to promote antibiotic resistance and, as a result, consequently, a higher recurrence of the disease. The goal of this study was to compare the ability of three MLST Clade 2 strains to form a biofilm in vitro: ICC-45 (ribotype SLO231/UK[CE]821), a ST41 toxinotype IXb isolated in Brazil; and two epidemic NAP1/027/ST01 strains: NAP1/027/ST01 (LIBA5756), isolated during a 2010 outbreak in Costa Rica and the reference epidemic strain NAP1/027/ST01 (R20291); and ATCC700057, a non-toxigenic strain. Methods: The ability of strains to form biofilm was evaluated using crystal violet staining. In addition, samples were stained with the Film Tracer biofilm matrix (Invitrogen®) and the biofilm matrix thickness was measured using confocal microscopy. The matrix architecture was determined using Scanning electron microscop. Confocal microscopy was used to detect the presence of toxin A (tcdA) using an anti-Clostridioides difficile TcdA antibody. The expression of virulence genes (tcdA, tcdB, tcdC, cdtB, spo0A, slpA, cwp66 and cwp84) was examined, as well as the effect of antibiotics metronidazole (MTZ) and vancomycin (VAN) on biofilm growth. Results: All of the strains tested formed a moderate biofilm with 1.1 3.5. After 72h, biofilm biomass of the NAP1/027/ST01 epidemic strains (LIBA5756 and R20291) was significantly higher than ICC-45 and ATCC 700057 biofilms, as confirmed by electron and confocal microscopy. At 120h, the LIBA5756 biofilm biomass decreased compared to other strains. The toxigenic strains R20291 or LIBA 5756 had higher expression of genes tcdA, tcdB, tcdC, cdtA, slpA and spo0A than ICC-45, but there were no significant differences in the expression levels of cdtB, cwp66 and cwp84. In epidemic strains, VAN and MTZ inhibited biofilm formation; however, in the ICC-45 strain, MIC concentrations of VAN and MIC and 4MIC of MTZ did not inhibit biofilm formation. Conclusion: The three MLST Clade 2 isolated from different rybotipes, two of which were isolated from Latin America, are competent biofilm-forming bacteria, indicating their ability to induce C. difficile infection recurrence, making treatment difficult.

Binding of the extracellular matrix laminin-1 to Clostridioides difficile strains

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.

Dietary Fiber Drives IL-1ß-Dependent Peritonitis Induced by Bacteroides fragilis via Activation of the NLRP3 Inflammasome.

Intestinal barrier is essential for dietary products and microbiota compartmentalization and therefore gut homeostasis. When this barrier is broken, cecal content overflows into the peritoneal cavity, leading to local and systemic robust inflammatory response, characterizing peritonitis and sepsis. It has been shown that IL-1ß contributes with inflammatory storm during peritonitis and sepsis and its inhibition has beneficial effects to the host. Therefore, we investigated the mechanisms underlying IL-1ß secretion using a widely adopted murine model of experimental peritonitis. The combined injection of sterile cecal content (SCC) and the gut commensal bacteria Bacteroides fragilis leads to IL-1ß-dependent peritonitis, which was mitigated in mice deficient in NLRP3 (nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3) inflammasome components. Typically acting as a damage signal, SCC, but not B. fragilis, activates canonical pathway of NLRP3 promoting IL-1ß secretion in vitro and in vivo. Strikingly, absence of fiber in the SCC drastically reduces IL-1ß production, whereas high-fiber SCC conversely increases this response in an NLRP3-dependent manner. In addition, NLRP3 was also required for IL-1ß production induced by purified dietary fiber in primed macrophages. Extending to the in vivo context, IL-1ß-dependent peritonitis was worsened in mice injected with B. fragilis and high-fiber SCC, whereas zero-fiber SCC ameliorates the pathology. Corroborating with the proinflammatory role of dietary fiber, IL-1R-deficient mice were protected from peritonitis induced by B. fragilis and particulate bran. Overall, our study highlights a function, previously unknown, for dietary fibers in fueling peritonitis through NLRP3 activation and IL-1ß secretion outside the gut.

MALDI-TOF MS: An alternative approach for ribotyping Clostridioides difficile isolates in Brazil.

Clostridioides difficile is an important organism causing healthcare-associated infections. It has been documented that specific strains caused multiple outbreaks globally, and patients infected with those strains are more likely to develop severe C. difficile infection (CDI). With the appearance of a variant strain, BI/NAP1 ribotype 027, responsible for several outbreaks and high mortality rates worldwide, the epidemiology of the CDI changed drastically in the United States, Europe, and some Latin American countries. Although the epidemic strain 027 was not yet detected in Brazil, there are ribotypes exclusively found in the country, such as, 131, 132, 133, 135, 142 and 143, which are responsible for outbreaks in Brazilian hospitals and nursing homes. Although PCR-ribotyping is the most used method in epidemiology studies of C. difficile, it is not available in Brazil. This study aimed to develop and validate an in-house database for detecting C. difficile ribotypes, usually involved in CDI in Brazilian hospitals, by using MALDI-TOF MS. A database with 19 different ribotypes, 13 with worldwide circulation and 6 Brazilian-restricted, was created based on 27 spectra readings of each ribotype. After BioNumerics analysis, neighbor-joining trees revealed that spectra were distributed in clusters according to ribotypes, showing that MALDI-TOF MS could discriminate all 19 ribotypes. Moreover, each ribotype showed a different profile with 42 biomarkers detected in total. Based on their intensity and occurrence, 13 biomarkers were chosen to compose ribotype-specific profiles, and in silico analysis showed that most of these biomarkers were uncharacterized proteins or well-conserved peptides, such as ribosomal proteins. A double-blind assessment using the 13 biomarkers correctly assigned the ribotype in 73% of the spectra analyzed, with 94%-100% of correct hits for 027 and for Brazilian ribotypes. Although further analyses are required, our results show that MALDI-TOF MS might be a reliable, fast and feasible alternative for epidemiological surveillance of C. difficile in Brazil.

Expression and antigenic analysis of the recombinant TRP36 protein from Ehrlichia canis São Paulo strain for serologic tests.

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.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry-based method for accurate discrimination of Staphylococcus schleiferi subspecies.

S. schleiferi is one of the main species isolated from canine otitis externa, pyoderma and from apparently healthy dogs. The species is divided into two subspecies, S. schleiferi schleiferi and S. schleiferi coagulans. MALDI-TOF MS does not distinguish correctly these two subspecies. This study aimed to identify biomarkers that could possibly discriminate Staphylococcus schleiferi subspecies by MALDI-TOF MS. Twelve strains (eight S. schleiferi schleiferi and four S. schleiferi coagulans) were firstly identified. Each isolate was submitted to a protein extraction protocol and subjected to spectrometry on Bruker Microflex LT mass spectrometer. Spectra were analyzed with the BioNumerics software v7.6. Our results showed that spectra clustered according to subspecies, and a set of five MALDI-TOF MS biomarkers were selected to enable the discrimination of S. schleiferi subspecies. In addition, these biomarkers were predicted to represent highly conserved proteins, which could contribute to the identification of subspecies-specific proteins that could be used for improved subspecies identification in clinical samples.

Identification of the alpha-enolase P46 in the extracellular membrane vesicles of Bacteroides fragilis

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.

Identification of the alpha-enolase P46 in the extracellular membrane vesicles of Bacteroides fragilis.

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.

Ribotypes associated with Clostridium difficile outbreaks in Brazil display distinct surface protein profiles.

Clostridium difficile is a spore-forming anaerobic intestinal pathogen that causes Clostridium difficile infection (CDI). C. difficile is the leading cause of toxin-mediated nosocomial antibiotic-associated diarrhea. The pathogenesis of CDI is attributed to two major virulence factors, TcdA and TcdB toxins, that cause the symptomatic infection. C. difficile also expresses a number of key proteins, including cell wall proteins (CWPs). S-layer proteins (SLPs) are CWPs that form a paracrystalline surface array that coats the surface of the bacterium. SLPs have a role in C. difficile binding to the gastrointestinal tract, but their importance in virulence need to be better elucidated. Here, we describe bottom-up proteomics analysis of surface-enriched proteins fractions obtained through glycine extraction of five C. difficile clinical isolates from Brazil using gel-based and gel-free approaches. We were able to identify approximately 250 proteins for each strain, among them SlpA, Cwp2, Cwp6, CwpV and Cwp84. Identified CWPs presented different amino acid coverage, which might suggest differences in post-translational modifications. Proteomic analysis of SLPs from ribotype 133, agent of C. difficile outbreaks in Brazil, revealed unique proteins and provided additional information towards in depth characterization of the strains causing CDI in Brazil.

Clostridium baratii: a rare case of pneumonia associated with an Alzheimer patient in Rio de Janeiro, Brazil.

INTRODUCTION: Clostridium baratii is rarely associated with human diseases. Infection is usuallcaused by ingestion of contaminated food, and infant botulism is the most common clinical presentation. CASE REPORT: Here we report a case of pneumonia by a non-toxigenic strain of C. baratii in an Alzheimer 70-year-old male with sepsis in Rio de Janeiro, Brazil. The micro-organism was identified by phenotypical tests, mass spectrometry (MALDI-TOF), DNA amplification (PCR) and sequencing of the 16S rRNA gene. Testing for the presence of botulinum F toxin was made using multiplex PCR. Bioassay for a large number of colonies was performed in mice to evaluate the production of any lethal toxin, but the results were negative. CONCLUSION: To our knowledge, there are no cases of C. baratii infection reported in Brazil and we highlight the importance of anaerobic lab tests in the standard routine of diagnosis.

Genetic diversity of Ehrlichia canis strains from naturally infected dogs in Rio de Janeiro, Brazil.

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 the 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.

Genetic diversity of Ehrlichia canis strains from naturally infected dogs in Rio de Janeiro, Brazil / Diversidade genética de cepas de Ehrlichia canis encontradas em cães naturalmente infectados no Rio de Janeiro, Brasil

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.

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 role of BmoR, a MarR Family Regulator, in the survival of Bacteroides fragilis during oxidative stress.

The intestinal opportunistic pathogen Bacteroides fragilis is among the most aerotolerant species of strict anaerobic bacteria and survives exposure to atmospheric oxygen for up to 72h. Under these circumstances, a strong oxygen stress response (OSR) mechanism is activated and the expression of as much as 45% of B. fragilis genes is altered. One of the most important regulators of this response is the product of the oxyR gene, but other regulation systems are in place during the OSR. The MarR family of transcriptional regulators has been shown to control several physiological events in bacteria, including response to stress conditions. In B. fragilis, at least three homologs of MarR regulators are present, one of which, bmoR, is upregulated during oxidative stress independently of oxyR. In this study, we demonstrate that the inactivation of the bmoR gene in B. fragilis diminishes its ability to withstand oxidative stress caused either by exposure to atmospheric oxygen or hydrogen peroxide. Recovery of growth rate on pre-oxidized media under anaerobiosis is slower than that observed in parental strain. Addition of hydrogen peroxide has a similar effect on the growth rate. Complementation of the mutant strain partially recovered the oxygen resistance phenotype, but the overexpression of the gene in the parental strain was also deleterious to a lesser extent. Our results indicate that BmoR has a role in the OSR in B. fragilis, particularly in the initial stages of oxygen exposure.

The Bfp60 surface adhesin is an extracellular matrix and plasminogen protein interacting in Bacteroides fragilis.

Plasminogen (Plg) is a highly abundant protein found in the plasma component of blood and is necessary for the degradation of fibrin, collagen, and other structural components of tissues. This fibrinolytic system is utilized by several pathogenic species of bacteria to manipulate the host plasminogen system and facilitate invasion of tissues during infection by modifying the activation of this process through the binding of Plg at their surface. Bacteroides fragilis is the most commonly isolated Gram-negative obligate anaerobe from human clinical infections, such as intra-abdominal abscesses and anaerobic bacteraemia. The ability of B. fragilis to convert plasminogen (Plg) into plasmin has been associated with an outer membrane protein named Bfp60. In this study, we characterized the function of Bfp60 protein in B. fragilis 638R by constructing the bfp60 defective strain and comparing its with that of the wild type regarding binding to laminin-1 (LMN-1) and activation of Plg into plasmin. Although the results showed in this study indicate that Bfp60 surface protein of B. fragilis is important for the recognition of LMN-1 and Plg activation, a significant slow activation of Plg into plasmin was observed in the mutant strain. For that reason, the possibility of another unidentified mechanism activating Plg is also present in B. fragilis cannot be discarded. The results demonstrate that Bfp60 protein is responsible for the recognition of laminin and Plg-plasmin activation. Although the importance of this protein is still unclear in the pathogenicity of the species, it is accepted that since other pathogenic bacteria use this mechanism to disseminate through the extracellular matrix during the infection, it should also contribute to the virulence of B. fragilis.