Priscila Soares Sabbadini

Pangenomic Study of Corynebacterium diphtheriae That Provides Insights into the Genomic Diversity of Pathogenic Isolates from Cases of Classical Diphtheria, Endocarditis, and Pneumonia

Corynebacterium diphtheriae is one of the most prominent human pathogens and the causative agent of the communicable disease diphtheria. The genomes of 12 strains isolated from patients with classical diphtheria, endocarditis, and pneumonia were completely sequenced and annotated. Including the genome of C. diphtheriae NCTC 13129, we herewith present a comprehensive comparative analysis of 13 strains and the first characterization of the pangenome of the species C. diphtheriae. Comparative genomics showed extensive synteny and revealed a core genome consisting of 1,632 conserved genes. The pangenome currently comprises 4,786 protein-coding regions and increases at an average of 65 unique genes per newly sequenced strain. Analysis of prophages carrying the diphtheria toxin gene tox revealed that the toxoid vaccine producer C. diphtheriae Park-Williams no. 8 has been lysogenized by two copies of the ω(tox)(+) phage, whereas C. diphtheriae 31A harbors a hitherto-unknown tox(+) corynephage. DNA binding sites of the tox-controlling regulator DtxR were detected by genome-wide motif searches. Comparative content analysis showed that the DtxR regulons exhibit marked differences due to gene gain, gene loss, partial gene deletion, and DtxR binding site depletion. Most predicted pathogenicity islands of C. diphtheriae revealed characteristics of horizontal gene transfer. The majority of these islands encode subunits of adhesive pili, which can play important roles in adhesion of C. diphtheriae to different host tissues. All sequenced isolates contain at least two pilus gene clusters. It appears that variation in the distributed genome is a common strategy of C. diphtheriae to establish differences in host-pathogen interactions.

Comparative analysis of two complete Corynebacterium ulcerans genomes and detection of candidate virulence factors.

BackgroundCorynebacterium ulcerans has been detected as a commensal in domestic and wild animals that may serve as reservoirs for zoonotic infections. During the last decade, the frequency and severity of human infections associated with C. ulcerans appear to be increasing in various countries. As the knowledge of genes contributing to the virulence of this bacterium was very limited, the complete genome sequences of two C. ulcerans strains detected in the metropolitan area of Rio de Janeiro were determined and characterized by comparative genomics: C. ulcerans 809 was initially isolated from an elderly woman with fatal pulmonary infection and C. ulcerans BR-AD22 was recovered from a nasal sample of an asymptomatic dog.ResultsThe circular chromosome of C. ulcerans 809 has a total size of 2,502,095 bp and encodes 2,182 predicted proteins, whereas the genome of C. ulcerans BR-AD22 is 104,279 bp larger and comprises 2,338 protein-coding regions. The minor difference in size of the two genomes is mainly caused by additional prophage-like elements in the C. ulcerans BR-AD22 chromosome. Both genomes show a highly similar order of orthologous coding regions; and both strains share a common set of 2,076 genes, demonstrating their very close relationship. A screening for prominent virulence factors revealed the presence of phospholipase D (Pld), neuraminidase H (NanH), endoglycosidase E (EndoE), and subunits of adhesive pili of the SpaDEF type that are encoded in both C. ulcerans genomes. The rbp gene coding for a putative ribosome-binding protein with striking structural similarity to Shiga-like toxins was additionally detected in the genome of the human isolate C. ulcerans 809.ConclusionsThe molecular data deduced from the complete genome sequences provides considerable knowledge of virulence factors in C. ulcerans that is increasingly recognized as an emerging pathogen. This bacterium is apparently equipped with a broad and varying set of virulence factors, including a novel type of a ribosome-binding protein. Whether the respective protein contributes to the severity of human infections (and a fatal outcome) remains to be elucidated by genetic experiments with defined bacterial mutants and host model systems.

Corynebacterium diphtheriae as an emerging pathogen in nephrostomy catheter-related infection: evaluation of traits associated with bacterial virulence

Corynebacterium diphtheriae still represents a global medical challenge, particularly due to the significant number of individuals susceptible to diphtheria and the emergence of non-toxigenic strains as the causative agents of invasive infections. In this study, we characterized the clinical and microbiological features of what we believe to be the first case of C. diphtheriae infection of a percutaneous nephrostomy catheter insertion site in an elderly patient with a fatal bladder cancer. Moreover, we demonstrated the potential role of adherence, biofilm formation and fibrin deposition traits in C. diphtheriae from the catheter-related infection. Non-toxigenic C. diphtheriae isolated from the purulent discharge (named strain BR-CAT5003748) was identified by the API Coryne system (code 1 010 324) and a multiplex PCR for detection of dtxR and tox genes. Strain BR-CAT5003748 showed resistance to oxacillin, ceftazidime and ciprofloxacin. In experiments performed in vitro, the catheter isolate was classified as moderately hydrophobic and as moderately adherent to polystyrene surfaces. Glass provided a more effective surface for biofilm formation than polystyrene. Micro-organisms adhered to (>1.5 x 10(6) c.f.u.) and multiplied on surfaces of polyurethane catheters. Microcolony formation (a hallmark of biofilm formation) and amorphous accretions were observed by scanning electron microscopy on both external and luminal catheter surfaces. Micro-organisms yielded simultaneous expression of localized adherence-like and aggregative-like (LAL/AAL) adherence patterns to HEp-2 cells. Interestingly, the coagulase tube test resulted in the formation of a thin layer of fibrin embedded in rabbit plasma by the non-toxigenic BR-CAT5003748 strain. In conclusion, C. diphtheriae should be recognized as a potential cause of catheter-related infections in at-risk populations such as elderly and cancer patients. LAL/AAL strains may be associated with virulence traits that enable C. diphtheriae to effectively produce biofilms on catheter surfaces. Biofilm formation and fibrin deposition could have contributed to the persistence of C. diphtheriae at the infected insertion site and the obstruction of the nephrostomy catheter.

Corynebacterium ulcerans diphtheria: an emerging zoonosis in Brazil and worldwide

The article is a literature review on the emergence of human infections caused by Corynebacterium ulcerans in many countries including Brazil. Articles in Medline/PubMed and SciELO databases published between 1926 and 2011 were reviewed, as well as articles and reports of the Brazilian Ministry of Health. It is presented a fast, cost-effective and easy to perform screening test for the presumptive diagnosis of C. ulcerans and C. diphtheriae infections in most Brazilian public and private laboratories. C. ulcerans spread in many countries and recent isolation of this pathogen in Rio de Janeiro, southeastern Brazil, is a warning to clinicians, veterinarians, and microbiologists on the occurrence of zoonotic diphtheria and C. ulcerans dissemination in urban and rural areas of Brazil and/or Latin America.O artigo revisa a literatura sobre a emergencia de infeccoes humanas causadas por Corynebacterium ulcerans em diversos paises, incluindo o Brasil. Foi realizada analise de artigos publicados entre 1926 e 2011 nas bases Medline/PubMed e SciELO, bem como artigos e informes do Ministerio da Saude. Apresenta-se um esquema de triagem, rapido, economico e de facil execucao, capaz de permitir a realizacao do diagnostico presuntivo de C. ulcerans e C. diphtheriae na maioria dos laboratorios brasileiros publicos e privados. A circulacao de C. ulcerans em varios paises, aliada aos recentes casos de isolamento do patogeno no Rio de Janeiro, e um alerta a clinicos, veterinarios e microbiologistas sobre a ocorrencia de difteria zoonotica e a circulacao do C. ulcerans em regioes urbanas e rurais do territorio nacional e/ou da America Latina.

Difteria pelo Corynebacterium ulcerans: uma zoonose emergente no Brasil e no mundo

The article is a literature review on the emergence of human infections caused by Corynebacterium ulcerans in many countries including Brazil. Articles in Medline/PubMed and SciELO databases published between 1926 and 2011 were reviewed, as well as articles and reports of the Brazilian Ministry of Health. It is presented a fast, cost-effective and easy to perform screening test for the presumptive diagnosis of C. ulcerans and C. diphtheriae infections in most Brazilian public and private laboratories. C. ulcerans spread in many countries and recent isolation of this pathogen in Rio de Janeiro, southeastern Brazil, is a warning to clinicians, veterinarians, and microbiologists on the occurrence of zoonotic diphtheria and C. ulcerans dissemination in urban and rural areas of Brazil and/or Latin America.O artigo revisa a literatura sobre a emergencia de infeccoes humanas causadas por Corynebacterium ulcerans em diversos paises, incluindo o Brasil. Foi realizada analise de artigos publicados entre 1926 e 2011 nas bases Medline/PubMed e SciELO, bem como artigos e informes do Ministerio da Saude. Apresenta-se um esquema de triagem, rapido, economico e de facil execucao, capaz de permitir a realizacao do diagnostico presuntivo de C. ulcerans e C. diphtheriae na maioria dos laboratorios brasileiros publicos e privados. A circulacao de C. ulcerans em varios paises, aliada aos recentes casos de isolamento do patogeno no Rio de Janeiro, e um alerta a clinicos, veterinarios e microbiologistas sobre a ocorrencia de difteria zoonotica e a circulacao do C. ulcerans em regioes urbanas e rurais do territorio nacional e/ou da America Latina.

Strain-dependent arthritogenic potential of the zoonotic pathogen Corynebacterium ulcerans.

During the last decade the majority of diphtheria cases in Europe had Corynebacterium ulcerans as the etiologic agent with dogs and cats as the reservoir hosts. However, little has been documented about the virulence factors of this zoonotic pathogen. To set up an in vivo experimental C. ulcerans infection model, conventional Swiss Webster mice were intravenously infected with different doses (from 1 × 10(7) to 5 × 10(9) bacteria per mouse) of C. ulcerans strains, namely 809 (from human lower respiratory tract), BR-AD22 (from asymptomatic dog nares) and CDC-KC279. Mortality rates were demonstrated by LD(50) values ranging from 1.9 × 10(8) to 1.3 × 10(9). Viable bacteria were recovered from blood, kidneys, liver, spleen and joints. For CDC-KC279 and 809 strains (2 × 10(8)mL(-1)) approximately 85% and 72% of animals with articular lesions were observed, respectively; BR-AD22-infected mice showed no signs of arthritis. CDC-KC279 and 809 strains exhibited higher arthritogenic potential when compared to the homologous toxigenic (ATCC27012) and non-toxigenic (ATCC27010) strains of Corynebacterium diphtheriae. A high number of affected joints and arthritis index in addition to the histopathological features, including subcutaneous edema, inflammatory infiltrate, damage to bone tissue and synoviocyte hypertrophy, indicated a strain-dependent ability of C. ulcerans strains to cause severe polyarthritis. A correlation between the arthritis index and systemic levels of IL-6 and TNF-α was observed for C. ulcerans strains, with the exception of the non-arthritogenic BR-AD22 strain. In conclusion, C. ulcerans revealed a strain-dependent arthritogenic potential independent of DNAse, PLD and diphtheria toxin production.

Non-opsonic phagocytosis of homologous non-toxigenic and toxigenic Corynebacterium diphtheriae strains by human U-937 macrophages

As interactions between bacteria and macrophages dictate the outcome of most infectious diseases, analyses of molecular mechanisms of non‐opsonic phagocytosis should lead to new approaches for the prevention of diphtheria and systemic Corynebacterium diphtheriae infections. The present study aimed to evaluate human macrophage–bacteria interactions in the absence of opsonin antibodies and the influence of the tox gene on this process. Homologous C. diphtheriae tox+ and tox– strains were evaluated for adhesion, entering and survival within U‐937 human macrophages at different incubation periods. Higher numbers of viable bacteria associated with and internalized by macrophages were demonstrated for the tox+ strain. However, viable intracellular bacteria were detected at T‐24 hr only for the tox– strain. Cytoskeletal inhibitors, cytochalasin E, genistein and colchicine, inhibited intracellular viability of both strains at different levels. Bacterial replication was evidenced at T‐24 hr in supernatants of monolayers infected with the tox– strain. Host cell death and nuclear alterations were evidenced by the Trypan blue exclusion assay and DAPI fluorescence microscopy. ELISA of histone‐associated DNA fragments allowed detection of apoptosis and necrosis induced by tox+ and tox– strains at T‐1 hr and T‐3 hr. In conclusion, human macrophages in the absence of opsonins may not be promptly effective at killing diphtheria bacilli. The presence of the tox gene influences the susceptibility of C. diphtheriae to human macrophages and the outcome of non‐opsonic phagocytosis. C. diphtheriae strains exhibit strategies to survive within macrophages and to exert apoptosis and necrosis in human phagocytic cells, independent of the tox gene.

Diphtheria outbreak in Maranhão, Brazil: microbiological, clinical and epidemiological aspects

We describe microbiological, clinical and epidemiological aspects of a diphtheria outbreak that occurred in Maranhão, Brazil. The majority of the 27 confirmed cases occurred in partially (n = 16) or completely (n = 10) immunized children (n = 26). Clinical signs and characteristic symptoms of diphtheria such as cervical lymphadenopathy and pseudomembrane formation were absent in 48% and 7% of the cases, respectively. Complications such as paralysis of lower limbs were observed. Three cases resulted in death, two of them in completely immunized children. Microbiological analysis identified the isolates as Corynebacterium diphtheriae biovar intermedius with a predominant PFGE type. Most of them were toxigenic and some showed a decrease in penicillin G susceptibility. In conclusion, diphtheria remains endemic in Brazil. Health professionals need to be aware of the possibility of atypical cases of C. diphtheriae infection, including pharyngitis without pseudomembrane formation.

Fibrinogen binds to nontoxigenic and toxigenic Corynebacterium diphtheriae strains

The production of fibrinous exudates may play an important role in determining the outcome of bacterial infection. Although pseudomembrane formation is a characteristic feature of diphtheria, little is known about the fibrinogen (Fbn)-binding properties of Corynebacterium diphtheriae strains and the influence of the gene that codes for diphtheria toxin (tox gene) in this process. In this study we demonstrated the ability of C. diphtheriae strains to bind to Fbn and to convert Fbn to fibrin. Bacterial interaction with rabbit plasma was evaluated by both slide and tube tests. Interaction of microorganisms with human Fbn was evaluated by both enzyme linked immunosorbent assay (ELISA) and fluorescein isothiocyanate-conjugated (FITC) Fbn binding assays. Nontoxigenic and toxigenic strains formed bacterial aggregates in the presence of plasma in the slide tests. The ability to convert Fbn to a loose web of fibrin in the plasma solution in the tube tests appeared to be a common characteristic of the species, including strains that do not carry the tox gene. Fbn binding to C. diphtheriae strains occurred at varying intensities, as demonstrated by the FITC-Fbn and ELISA binding assays. Our data suggest that the capacity to bind to Fbn and to convert Fbn to fibrin may play a role in pseudomembrane formation and act as virulence determinants of both nontoxigenic and toxigenic strains.

SubMICs of penicillin and erythromycin enhance biofilm formation and hydrophobicity of Corynebacterium diphtheriae strains.

Subinhibitory concentrations (subMICs) of antibiotics may alter bacterial surface properties and change microbial physiology. This study aimed to investigate the effect of a subMIC (⅛ MIC) of penicillin (PEN) and erythromycin (ERY) on bacterial morphology, haemagglutinating activity, cell-surface hydrophobicity (CSH) and biofilm formation on glass and polystyrene surfaces, as well as the distribution of cell-surface acidic anionic residues of Corynebacterium diphtheriae strains (HC01 tox(-) strain; CDC-E8392 and 241 tox(+) strains). All micro-organisms tested were susceptible to PEN and ERY. Growth in the presence of PEN induced bacterial filamentation, whereas subMIC of ERY caused cell-size reduction of strains 241 and CDC-E8392. Adherence to human erythrocytes was reduced after growth in the presence of ERY, while CSH was increased by a subMIC of both antibiotics in bacterial adherence to n-hexadecane assays. Conversely, antibiotic inhibition of biofilm formation was not observed. All strains enhanced biofilm formation on glass after treatment with ERY, while only strain 241 increased glass adherence after cultivation in the presence of PEN. Biofilm production on polystyrene surfaces was improved by ⅛ MIC of ERY. After growth in the presence of both antimicrobial agents, strains 241 and CDC-E8392 exhibited anionic surface charges with focal distribution. In conclusion, subMICs of PEN and ERY modified bacterial surface properties and enhanced not only biofilm formation but also cell-surface hydrophobicity. Antibiotic-induced biofilm formation may contribute to the inconsistent success of antimicrobial therapy for C. diphtheriae infections.