Comparative fingerprinting analysis of Campylobacter jejuni subsp. jejuni strains by amplified-fragment length polymorphism genotyping (original) (raw)
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Discrimination of Campylobacter jejuni isolates by fla gene sequencing
Journal of Clinical Microbiology, 1997
Comparison of the entire coding sequence of flaA (1,764 nucleotides) from 15 isolates of Campylobacter jejuni showed two regions of high variability, one region approximately from base positions 700 to 1,450 and a short variable region (SVR) from base positions 450 to 600. Parsimony analysis of the SVR sequences yielded a dendrogram similar to that which was derived by analysis of the entire gene. PCR was used to generate templates, and the SVR was sequenced with primers constructed to hybridize to conserved flanking sequences. The SVRs of 22 isolates of C. jejuni from four outbreaks that have been well characterized and a larger panel of isolates from three additional outbreaks were sequenced. Analysis of the nucleotide sequences produced results that grouped the isolates very similarly to other subtyping techniques. Sequence data were also generated for isolates from three additional outbreaks. Categorizing the isolates by fla SVR DNA sequence placed them in epidemiologically rele...
Journal of Clinical Microbiology, 2002
A molecular typing approach for Campylobacter jejuni and Campylobacter coli was developed with restriction fragment length polymorphism analysis of a 9.6-kb PCR-amplified portion of the lipopolysaccharide gene cluster. Sixty-one Penner serotype reference strains were analyzed with this new genotyping scheme, and 32 genogroups were found. Eleven additional genogroups were obtained from 87 clinical C. jejuni strains tested. This molecular typing method shows a correlation with the Penner heat-stable serotyping method, a phenotypic typing method based on lipopolysaccharide structures that is often used as a "gold standard" for subtyping Campylobacter spp. This strong correlation suggests that the data obtained can be directly compared with epidemiological data collected in the past by classical serotyping of C. jejuni and C. coli. In contrast to the high percentage of nontypeability by phenotyping, this molecular typing method results in 100% typeability and provides a superior alternative to serotyping.
Journal of Medical Microbiology, 2006
This investigation describes the development of a generally applicable, bioinformatics-driven, single-nucleotide polymorphism (SNP) genotyping assay for the common bacterial gastrointestinal pathogen Campylobacter jejuni. SNPs were identified in silico using the program 'Minimum SNPs', which selects for polymorphisms providing the greatest resolution of bacterial populations based on Simpson's index of diversity (D). The high-D SNPs identified in this study were derived from the combined C. jejuni/Campylobacter coli multilocus sequence typing (MLST) database. Seven SNPs were found that provided a D of 0?98 compared with full MLST characterization, based on 959 sequence types (STs). The seven high-D SNPs were interrogated using allele-specific real-time PCR (AS kinetic PCR), which negates the need for expensive labelled primers or probes and requires minimal assay optimization. The total turnaround time of the SNP typing assay was approximately 2 h. Concurrently, 69 C. jejuni isolates were subjected to MLST and flagellin A short variable region (flaA SVR) sequencing and combined with a population of 84 C. jejuni and C. coli isolates previously characterized by these methods. Within this collection of 153 isolates, 19 flaA SVR types (D=0?857) were identified, compared with 40 different STs (D=0?939). When MLST and flaA SVR sequencing were used in combination, the discriminatory power was increased to 0?959. In comparison, SNP typing of the 153 isolates alone provided a D of 0?920 and was unable to resolve a small number of unrelated isolates. However, addition of the flaA SVR locus to the SNP typing procedure increased the resolving power to 0?952 and clustered isolates similarly to MLST/flaA SVR. This investigation has shown that a seven-member C. jejuni SNP typing assay, used in combination with sequencing of the flaA SVR, efficiently discriminates C. jejuni isolates.
Journal of Clinical Microbiology, 2012
Campylobacter spp. are a leading cause of bacterial gastroenteritis worldwide. The need for molecular subtyping methods with enhanced discrimination in the context of surveillance-and outbreak-based epidemiologic investigations of Campylobacter spp. is critical to our understanding of sources and routes of transmission and the development of mitigation strategies to reduce the incidence of campylobacteriosis. We describe the development and validation of a rapid and high-resolution comparative genomic fingerprinting (CGF) method for C. jejuni. A total of 412 isolates from agricultural, environmental, retail, and human clinical sources obtained from the Canadian national integrated enteric pathogen surveillance program (C-EnterNet) were analyzed using a 40-gene assay (CGF40) and multilocus sequence typing (MLST). The significantly higher Simpson's index of diversity (ID) obtained with CGF40 (ID ؍ 0.994) suggests that it has a higher discriminatory power than MLST at both the level of clonal complex (ID ؍ 0.873) and sequence type (ID ؍ 0.935). High Wallace coefficients obtained when CGF40 was used as the primary typing method suggest that CGF and MLST are highly concordant, and we show that isolates with identical MLST profiles are comprised of isolates with distinct but highly similar CGF profiles. The high concordance with MLST coupled with the ability to discriminate between closely related isolates suggests that CFG40 is useful in differentiating highly prevalent sequence types, such as ST21 and ST45. CGF40 is a high-resolution comparative genomics-based method for C. jejuni subtyping with high discriminatory power that is also rapid, low cost, and easily deployable for routine epidemiologic surveillance and outbreak investigations.
Diagnostic Microbiology and Infectious Disease, 1996
Seventeen sporadic Campylobacter jejuni enteritis cases occurred in Taichung City, Taiwan between July 1995 and September 2995. Pulsed-field gel electrophoresis (PFGE) and enterobacterial repetitive intergenic consensus (ERIC-Z) primed polymerase chain reaction (PCR) techniques were compared for the epidemiological typing of the 17 C. jejuni isolates. Fourteen distinct PFGE fingerprint patterns were observed. Fifteen distinct PCR fingerprint patterns were demonstrated. Two clusters of isolates (isolates 5 and 6; isolates 10, II respec-tively) were found to be genetically indistinguishable by both methods. In conclusion, we consider that PFGE is a highly reproducible method for determining the relatedness among the C. jejuni isolates in this study, although their limited numbers of restriction fragments may reduce the discriminatory power. Although less reproducible than PFGE typing, ERIC-l primed PCR can be used as a simple and rapid tool to discriminate different strains of C. jejuni. 0 1997 Elsevier Science Inc.
Journal of Microbiological Methods, 2003
Campylobacter jejuni is a frequent cause of bacterial gastroenteritis in humans all over the world. Several molecular typing methods are used to study the epidemiology of Campylobacter spp. infections. The aim of the present study was to investigate the application of single-strand conformation polymorphism (SSCP) and denaturing gradient gel electrophoresis (DGGE) analysis as rapid primary subtyping methods for C. jejuni. A variable fragment from the 3Vend of the flaA to the 3Vend of the intergenic region, separating the flaA and flaB genes, was subjected to SSCP and DGGE analysis. A total of 48 clinical C. jejuni isolates, 49 C. jejuni strains isolated from poultry, 2 strains isolated from ducks and 1 strain isolated from a pheasant were assigned to 24 distinct SSCP patterns. Sequence analysis of the respective DNA fragments revealed that every different fla sequence type could be distinguished by SSCP. DGGE proved to be equally discriminatory. Both methods can be applied as primary subtyping methods, because pulsed-field gel electrophoresis (PFGE) and amplified fragment length polymorphism (AFLP) analysis further differentiated isolates belonging to the same fla sequence types. D
Journal of Applied Microbiology, 2004
Aims: To use amplified fragment length polymorphism (AFLP) analysis to evaluate the genetic relatedness among 254 Campylobacter jejuni reference and field strains of diverse origin representing all defined ÔPennerÕ serotypes for this species. Methods and Results: Field strains (n ¼ 207) from human diarrhoea and diverse animal and environmental sources were collected mainly through a National surveillance programme in Denmark and serotyped by use of the established ÔPennerÕ scheme. Genetic relationships among these isolates, and the archetypal serotype reference strains, were assessed by numerical analysis of AFLP profiles derived from genomic DNA. Extensive genetic diversity was seen among the strains examined; however, 43 groups of isolates were identified at the 92% similarity (S-) level. Thirteen groups contained isolates from a single host, possibly representing genotypes of Ôlow riskÕ to human health. The remaining 30 groups contained isolates from humans, chickens and associated food products, cattle, sheep, turkeys, ostriches and/or dogs. Strains assigned to serotypes 2, 6/7, 11 and 12 formed major clusters at the 77AE6% S-level. Most other serotypes did not form homogeneous clusters. Conclusions: High-resolution genotyping applied to strains from a comprehensive range of sources provides evidence for multiple sources of sporadic C. jejuni infection. The results suggest that public health protection measures should be directed at all foods of animal origin. Significance and Impact of the Study: The genetic relatedness among all ÔPennerÕ serotypes of C. jejuni is assessed by AFLP analysis. In addition, further evidence of epidemic and host-specific clones of C. jejuni is provided.
Foodborne Pathogens and Disease, 2009
Comparative genome indexing (CGI) using whole-genome DNA microarrays was evaluated as a means of genotyping Campylobacter jejuni relative to two standard methods, pulsed-field gel electrophoresis (PFGE) and flaA short variable region sequencing (flaA SVR typing). Thirty-six geographically diverse C. jejuni isolates were selected from a collection of cattle and chicken isolates. The BioNumerics Ò software program was used for cluster analysis of the data from all 36 isolates for each of the three typing methods. Comparative genome indexing assigned a unique type to each isolate while PFGE and flaA SVR distinguished 29 and 35 different types, respectively. The four common types identified by PFGE were also closely related by CGI, and the overall similarity of the CGI results to those for PFGE indicates the value of CGI as a more informative alternative to PFGE. While flaA SVR was very discriminative, the isolates were all highly similar (>78%) resulting in finer distinctions between isolates and fewer genotypic relations to CGI or PFGE. Campylobacter jejuni is one of the most common causative agents of bacterial gastroenteritis in the world. The development of CGI as a molecular typing tool for C. jejuni offers a highly effective and informative means of further understanding the epidemiology of this ubiquitous pathogen.
Repetitive Element Sequence-Based PCR Typing for Improved Discrimination of Campylobacter Jejuni
The aim of our studies was to evaluate rep-PCR typing using different primers as a fast genotyping method for discrimination of C. jejuni. Also a microfluidic electrophoretic DNA separation method for rapid DNA fingerprinting based on Lab-on-a-Chip technology was tested. Based on the test results, the single polytrinucleotide (GTG)5 was chosen as primer. Nine strains representing three epidemiological groups from the CAMPYNET network strains were assigned to three different groups by both (GTG)5-PCR and PFGE. Typing of 72 epidemiologically independent strains using (GTG)5-PCR and PFGE clustered the strains in 60 and 53 clusters, respectively, with an identical discriminatory power (D=0,99). When PCR-products were separated on the Agilent 2100 Bioanalyzer Lab-on-a-Chip device the strains were grouped with the same discriminatory power, though some strains were clustered differently compare to gel-based DNA amplicon separation. The main disadvantage of (GTG)5-PCR typing was difference...