RESUMO
A focused oligonucleotide microarray featuring 62 probes targeting strain variable regions of the Clostridium botulinum strain ATCC 3502 genome sequence was developed to differentiate C. botulinum type A strains. The strain variable regions were selected from deletions identified among a panel of 10 type A strains compared to the strain ATCC 3502 genome sequence using high density comparative genomic hybridization microarrays. The focused microarray also featured specific probes for the detection of the neurotoxin genes of various serotypes (A-G), toxin gene cluster components (ha70 and orfX1), and fldB as a marker for proteolytic clostridia (Group I). Eight pairs of strains selected from separate type A botulism outbreaks were included in the 27 subtype A1-A4 strains examined in this study. Each outbreak related strain pair consisted of strains isolated from different sources (stool and food). Of the eight outbreak related strain pairs, six groups of strains with indistinguishable hybridization patterns were identified. Outbreak related strains were shown to have identical hybridization patterns. Strain pairs from three separate outbreaks involving strains harboring both the type A neurotoxin gene (bont/A) and an unexpressed type B neurotoxin gene (bont/B) shared the same probe hybridization profile. The focused microarray format provides a rapid approach for neurotoxin gene detection and preliminary determination of the relatedness of strains isolated from different sources.
Assuntos
Proteínas de Bactérias/genética , Clostridium botulinum tipo A/genética , Hibridização Genômica Comparativa/métodos , Neurotoxinas/genética , Botulismo/microbiologia , Clostridium botulinum tipo A/classificação , Clostridium botulinum tipo A/isolamento & purificação , Sondas de DNA/genética , DNA Bacteriano/química , DNA Bacteriano/genética , Humanos , Análise em Microsséries/métodos , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , Especificidade da EspécieRESUMO
A group of five clonally related Clostridium botulinum type A strains isolated from different sources over a period of nearly 40 years harbored several conserved genetic properties. These strains contained a variant bont/A1 with five nucleotide polymorphisms compared to the gene in C. botulinum strain ATCC 3502. The strains also had a common toxin gene cluster composition (ha-/orfX+) similar to that associated with bont/A in type A strains containing an unexpressed bont/B [termed A(B) strains]. However, bont/B was not identified in the strains examined. Comparative genomic hybridization demonstrated identical genomic content among the strains relative to C. botulinum strain ATCC 3502. In addition, microarray data demonstrated the absence of several genes flanking the toxin gene cluster among the ha-/orfX+ A1 strains, suggesting the presence of genomic rearrangements with respect to this region compared to the C. botulinum ATCC 3502 strain. All five strains were shown to have identical flaA variable region nucleotide sequences. The pulsed-field gel electrophoresis patterns of the strains were indistinguishable when digested with SmaI, and a shift in the size of at least one band was observed in a single strain when digested with XhoI. These results demonstrate surprising genomic homogeneity among a cluster of unique C. botulinum type A strains of diverse origin.
