Informe especial: Actualización epidemiológica de difteria / Special report: Epidemiological update of diphtheria

La difteria es una enfermedad infecciosa grave, con potencial epidémico, para la que existe una vacuna eficaz. Es una infección bacteriana aguda causada por la liberación de una exotoxina específica, producida por el bacilo gram positivo, Corynebacterium diphtheriae. Sólo las cepas toxigénicas causan enfermedad. La enfermedad se presenta principalmente en menores de 15 años de edad sin vacunación o con vacunación incompleta; no obstante puede presentarse en adultos en áreas con baja cobertura de vacunación. En este informe se presentan distintos aspectos de la enfermedad y la situación epidemiológica en las Américas y en Argentina: definiciones de caso, medidas de prevención y de control

Construção, clonagem e expressão do fragmento B da toxina diftérica de Corynebacterium diphtheriae (cepa PW- 8) em Mycobacterium bovis BCG sub-cepa Moreau / Construction, cloning and expression of the fragment B of diphtheria toxin from Corynebacterium diphtheriae (strain PW-8) in Mycobacterium bovis BCG Moreau sub-strain

A vacina anti-diftérica de uso corrente no Brasil (DTP), embora de alta eficácia na prevenção da difteria, está associada com episódios de toxicidade e reatogenicidade no recipiente vacinal, resultantes de proteínas residuais derivadas do processo de produção ou detoxificação. Estratégias para o desenvolvimento de vacinas menos reatogênicas e ao mesmo tempo mais eficazes e economicamente viáveis contra a difteria têm sido alvo de intensa investigação. A alternativa proposta por nosso grupo é a utilização da vacina contra a tuberculose (Mycobacterium bovis BCG sub-cepa Moreau), como vetor do gene que codifica o fragmento B da toxina diftérica (dtb) de 58,3 kDa. Neste trabalho o dtb foi clonado no vetor micobacteriano bifuncional (pUS977) de expressão citoplasmática e os clones recombinantes (pUS977dtbPW8), após a transformação do BCG, foram testados com relação a expressão do DTB em BCG e quanto a antigenicidade frente a anticorpos policlonais anti-toxóide diftérico por Immunobloting. A integridade do gene dtb e a identidade das sequências de DNA da construção plasmidial pUS977dtbPW8 foram confirmadas por sequenciamento de DNA e análise de similaridade. A imunogenicidade do BCGr pUS977dtbPW8 expressando o DTB foi investigada em camundongos BALB/c, os resultados obtidos revelaram uma soroconversão específica (IgG). A infectividade e atividade microbicida do BCGr pUS977dtbPW8 no ambiente intracelular foi avaliada através da infecção de linhagens de células de monócitos humano (THP-1), os dados obtidos indicaram que houve sobrevivência intracelular em até 12 dias. Nesse contexto, esplenócitos dos camundongos imunizados com 30 e 60 dias foram extraídos, mostrando que o BCGr pUS977dtbPW8 persistiu até 60 dias na ausência de pressão seletiva e a viabilidade celular não sofreu alteração significativa durante o período testado...

The diphtheria vaccine currently used in Brazil (DTP), despite its history of high efficacy in the prevention of diphtheria, is associated with episodes of toxicity and vaccine reactogenicity in the vaccinee, resulting from the presence in the vaccine of residual proteins derived from the production process or detoxification. Strategies for the development of new vaccines more effective and economically viable against diphtheria have been the subject of intense investigation. The alternative proposed by our group is the use of the vaccine against tuberculosis (Mycobacterium bovis BCG Moreau sub strain) as a vector for the gene that encodes the 58.3 kDa fragment B of the diphtheria toxin (DTB). In our project the dtb gene was cloned into the bifunctional vector pUS977 for cytoplasmic expression and recombinant BCG (rBCG) clones, selected after transformation of BCG, were tested for expression of the DTB polypeptide and antigenicity against polyclonal antibodies anti- diphtheria toxoid by immunoblotting. The integrity and identity of the DNA sequence encoding the dtb gene carried by the plasmid construct pUS977dtbPW8 was confirmed by DNA Sequencing and Analysis of Similarity. The immunogenicity of the rBCG expressing the DTB was investigated in BALB/c mice and the results revealed a specific seroconversion (IgG)...

Genomic library screening for viruses from the human dental plaque revealed pathogen-specific lytic phage sequences.

Bacterial pathogenesis presents an astounding arsenal of virulence factors that allow them to conquer many different niches throughout the course of infection. Principally fascinating is the fact that some bacterial species are able to induce different diseases by expression of different combinations of virulence factors. Nevertheless, studies aiming at screening for the presence of bacteriophages in humans have been limited. Such screening procedures would eventually lead to identification of phage-encoded properties that impart increased bacterial fitness and/or virulence in a particular niche, and hence, would potentially be used to reverse the course of bacterial infections. As the human oral cavity represents a rich and dynamic ecosystem for several upper respiratory tract pathogens. However, little is known about virus diversity in human dental plaque which is an important reservoir. We applied the culture-independent approach to characterize virus diversity in human dental plaque making a library from a virus DNA fraction amplified using a multiple displacement method and sequenced 80 clones. The resulting sequence showed 44% significant identities to GenBank databases by TBLASTX analysis. TBLAST homology comparisons showed that 66% was viral; 18% eukarya; 10% bacterial; 6% mobile elements. These sequences were sorted into 6 contigs and 45 single sequences in which 4 contigs and a single sequence showed significant identity to a small region of a putative prophage in the Corynebacterium diphtheria genome. These findings interestingly highlight the uniqueness of over half of the sequences, whilst the dominance of a pathogen-specific prophage sequences imply their role in virulence.

Bacteriophage-based vectors for site-specific insertion of DNA in the chromosome of Corynebacteria.

In Corynebacterium diphtheriae, diphtheria toxin is encoded by the tox gene of some temperate corynephages such as beta. beta-like corynephages are capable of inserting into the C. diphtheriae chromosome at two specific sites, attB1 and attB2. Transcription of the phage-encoded tox gene, and many chromosomally encoded genes, is regulated by the DtxR protein in response to Fe(2+) levels. Characterizing DtxR-dependent gene regulation is pivotal in understanding diphtheria pathogenesis and mechanisms of iron-dependent gene expression; although this has been hampered by a lack of molecular genetic tools in C. diphtheriae and related Coryneform species. To expand the systems for genetic manipulation of C. diphtheriae, we constructed plasmid vectors capable of integrating into the chromosome. These plasmids contain the beta-encoded attP site and the DIP0182 integrase gene of C. diphtheriae NCTC13129. When these vectors were delivered to the cytoplasm of non-lysogenic C. diphtheriae, they integrated into either the attB1 or attB2 sites with comparable frequency. Lysogens were also transformed with these vectors, by virtue of the second attB site. An integrated vector carrying an intact dtxR gene complemented the mutant phenotypes of a C. diphtheriae DeltadtxR strain. Additionally, strains of beta-susceptible C. ulcerans, and C. glutamicum, a species non-permissive for beta, were each transformed with these vectors. This work significantly extends the tools available for targeted transformation of both pathogenic and non-pathogenic Corynebacterium species.

Characterization of bacteriophages from tox-containing, non-toxigenic isolates of Corynebacterium diphtheriae.

Non-toxigenic strains of Corynebacterium diphtheriae continue to cause disease within immunized populations. A subset of these corynebacteria carry the diphtheria toxin gene but in a cryptic form. To determine whether such strains might contribute to the re-emergence of functional toxin genes, the phages and tox mutations within three clone types were examined. tox-containing, beta-related phages were isolated from two of the strain types. The third isolate appeared to harbour a defective prophage. One of the tox- phages encoded truncated, yet enzymatically-active, forms of diphtheria toxin, suggesting that it had sustained a point mutation within the latter half of its toxin gene. In contrast, the other mutant phage did not elicit the production of either a cross-reacting material or an ADP-ribosylating activity. Complementation tests employing a series of double lysogens confirmed that the mutations responsible for the non-toxigenic phenotype of all of the phages were cis dominant. Given these findings, it is reasonable to hypothesize that tox+ genes can arise within human populations by either homologous recombination between two distinct tox- phages or spontaneous reversion within a single mutant allele.

Extended host range of a beta-related corynebacteriophage.

Unlike most beta-related phages isolated from Corynebacterium diphtheriae, phage 782 readily plaqued on strains of C. ulcerans and C. pseudotuberculosis. The extended host range of phage 782 was not, however, due to a greater ability of the phage to absorb to bacterial surfaces. Using chemical mutagenesis, a number of phage mutants were isolated which had diminished capacities to infect C. ulcerans, suggesting the existence of a locus (ehr) for extended host range. Pre-lysogenization of C. ulcerans strains with phage 782, but not its mutant form or other beta-related phages, rendered them susceptible to infection by previously excluded phages. An examination of recombinant between phages 782, pie, and beta localized ehr to a 7 kilobase region of DNA including attP. The data are compatible with the notion that ehr encodes an anti-restriction function.