Analysis of Neurotoxin Cluster Genes in Clostridium botulinum Strains Producing Botulinum Neurotoxin Serotype A Subtypes
Journal - Applied and Environmental Microbiology
Neurotoxin cluster gene sequences and arrangements were elucidated for strains of Clostridium botulinum encoding botulinum neurotoxin (BoNT) subtypes A3, A4, and a unique A1-producing strain (HA- Orfx+ A1). These sequences were compared to the known neurotoxin cluster sequences of C. botulinum strains that produce BoNT/A1 and BoNT/A2 and possess either a hemagglutinin (HA) or an Orfx cluster, respectively. The A3 and HA- Orfx+ A1 strains demonstrated a neurotoxin cluster arrangement similar to that found in A2. The A4 strain analyzed possessed two sets of neurotoxin clusters that were similar to what has been found in the A(B) strains: an HA cluster associated with the BoNT/B gene and an Orfx cluster associated with the BoNT/A4 gene. The nucleotide and amino acid sequences of the neurotoxin cluster-specific genes were determined for each neurotoxin cluster and compared among strains. Additionally, the ntnh gene of each strain was compared on both the nucleotide and amino acid levels. The degree of similarity of the sequences of the ntnh genes and corresponding amino acid sequences correlated with the neurotoxin cluster type to which the ntnh gene was assigned.
Genetic Homogeneity of Clostridium botulinum Type A1 Strains with Unique Toxin Gene Clusters†
Journal - Applied and Environmental Microbiology
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.
Microarray-based detection of genetic heterogeneity, antimicrobial resistance, and the viable but nonculturable state in human pathogenic Vibrio spp.
Journal - PNAS
Accepted for publication November 8, 2005.
The morbidity and mortality associated with Vibrio-mediatedwaterborne diseases necessitates the development of sensitivedetection technologies that are able to elucidate the identity,potential pathogenicity, susceptibility, and viability of contaminatingbacteria in a timely manner. For this purpose, we have designeda single multiplex PCR assay to simultaneously amplify 95 diagnosticregions (encompassing species/serogroup-specific, antimicrobialresistance, and known toxin markers) and combined it with along oligonucleotide microarray to create a platform capableof rapidly detecting and discriminating the major human pathogenicspecies from the genus Vibrio: V. cholerae, V. parahaemolyticus,V. vulnificus, and V. mimicus. We were able to validate thisstrategy by testing 100 geographically and temporally distributedisolates and observed an excellent concordance between species-and serotype-level microarray-based identification and traditionaltyping methods. In addition to accurate identification, themicroarray simultaneously provided evidence of antibiotic resistancegenes and mobile genetic elements, such as sulfamethoxazole-trimethoprimconstins and class I integrons, and common toxin (ctxAB, rtxA,hap, hlyA, tl, tdh, trh, vvhA, vlly, and vmhA) and pathogenicity(tcpA, type III secretion system) genes that are associatedwith pathogenic Vibrio. The versatility of this method was furtherunderscored by its ability to detect the expression of knowntoxin and virulence genes from potentially harmful viable butnonculturable organisms. The results suggest that this molecularidentification method provides rapid and definitive informationthat would be of value in epidemiological, environmental, andhealth risk assessment surveillance.
Conflict of interest statement: No conflicts declared.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: VBNC, viable but nonculturable; CT, cholera toxin;SXT, sulfamethoxazoletrimethoprim; ASW, artificial seawater;TTSS, type III secretion system; VPI-2, Vibrio pathogenicityisland-2.© 2005 by The National Academy of Sciences of the USA
|Keywords : ||pathogen detection • molecular diagnostics • cholera|