Molecular Evidence for Dissemination of Unique Campylobacter jejuni Clones in Curacao, Netherlands Antilles (original) (raw)
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The application of genotyping techniques to the epidemiological analysis of Campylobacter jejuni
Epidemiology and Infection, 1996
SummaryCampylobacter jejuniserogroup reference strains and collections of sporadic and outbreak- associated isolates were examined for restriction fragment length polymorphisms (RFLPs), usingC. jejunirandom chromosomal and 16S rRNA gene probes. A collection of 48 Penner (HS) and 14 Lior (HL) serogroup reference strains, plus 10 clinical isolates, generated 35 RFLP and 26 ribotype patterns. In combination the two loci generated 48 distinct genotypes. Both probes were able to differentiate between certain random isolates of the same HS/HL serogroups but greater discrimination was obtained with RFLP than with ribotyping. Genotyping distinguished accurately between related and unrelated strains when applied to several outbreaks. Genotypic analysis ofC. jejuniby restriction fragment length polymorphisms is a valuable technique for epidemiological typing. Chromosomal variation detected by the two unlinked probe loci provides some information about the genetic relationship between isolates.
Journal of clinical microbiology, 2000
For epidemiological tracing of the thermotolerant Campylobacter species C. jejuni and C. coli, reliable and highly discriminatory typing techniques are necessary. In this study the genotyping techniques of flagellin typing (flaA typing), pulsed-field gel electrophoresis (PFGE), automated ribotyping, and amplified fragment length polymorphism (AFLP) fingerprinting were compared. The following aspects were compared: computer-assisted analysis, discriminatory power, and use for epidemiological typing of campylobacters. A set of 50 campylobacter poultry isolates from The Netherlands and neighboring countries was analyzed. Computer-assisted analysis made cluster analysis possible and eased the designation of different genotypes. AFLP fingerprinting was the most discriminatory technique, identifying 41 distinct genotypes, while PFGE identified 38 different types, flaA typing discriminated 31 different types, and ribotyping discriminated 26 different types. Furthermore, AFLP analysis was t...
Genotyping of Human Campylobacter jejuni Isolates in Greece by Pulsed-Field Gel Electrophoresis
Molecular Diagnosis & Therapy, 2006
Background: Pulsed-field gel electrophoresis (PFGE) typing has been recognized by several groups as a Abstract relatively simple and quick method for genotyping of Campylobacter jejuni (C. jejuni). The present study was carried out to determine the genetic variations among clinical isolates of C. jejuni from Greece and to establish a database, which could be used for future epidemiological and clinical studies. Methods: A total of 93 C. jejuni clinical isolates of known flagellin subunit A (flaA) genotype, serotype, and antimicrobial susceptibility pattern, were collected from a general hospital in the Attica region of Greece, between the years 2000 and 2003. The PFGE profiles of SmaI DNA digests of each strain were compared using a bin analysis based on 44 molecular size intervals. Results: Forty-three different PFGE types, designated as C. jejuni (C. j.) 1 Greece (GR) to C. j. 43 GR, were identified. There was no statistically significant association of PFGE type with flaA genotype, serotype, or antimicrobial susceptibility pattern. However, PFGE typing did show a remarkable discriminatory ability within the non-serotypable group. Conclusion: Evaluating our results, we observed that (i) there was no statistically significant clonality of a certain PFGE type among the strains examined, and (ii) the discriminatory ability of PFGE typing was much better than that of the other typing methods. This is the first report of the use of bin patterns to compare the PFGE genotypes identified.
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 Clinical Microbiology, 2001
The Agilent 2100 bioanalyzer (Agilent Technologies, Palo Alto, Calif.) utilizes capillary electrophoresis on a microchip device (LabChip 7500; Caliper Technologies, Mountain View, Calif.) that is capable of rapidly sizing small DNA fragments. To determine whether the system could replace conventional restriction fragment length polymorphism (RFLP) typing by agarose gel electrophoresis, we compared the analyzer with conventional flagellin RFLP for typing Campylobacter jejuni. Ninety-seven isolates representing 46 Fla types were initially analyzed. Correct Fla types were detected in 59% of the isolates. The major problem with the system was in resolving samples containing multiple DNA fragments differing from 8 to 20 bp. Overall, the bioanalyzer has the potential to replace conventional RFLP analysis by gel electrophoresis, but improvements in the chip separation are needed.
Journal of Medical Microbiology, 2001
Pulsed-®eld gel electrophoresis (PFGE) was used to analyse 147 isolates collected in two regions of Que Âbec province (Estrie and Montre Âal) between March 1998 and Feb. 1999, to determine the utility of molecular strain typing for a population-based collection of Campylobacter jejuni and to compare directly the discriminatory power of Sma I and Kpn I restriction digests. With a combination of epidemiological criteria including space and time plus molecular strain typing, 49% of isolates from Estrie and 39% of isolates from Montre Âal were identi®ed as belonging to a putative cluster. For 41% of the cases, sources were either missing or explicitly unknown; the remaining sources were subject to recall bias. Thus, the evaluation of sporadic cases of campylobacter enteritis by descriptive clinical investigation alone is neither sensitive nor reliable for identifying sources of infection. In the PFGE analysis, Kpn I digests provided appreciably greater discriminatory power than Sma I digests. When combining the PFGE analyses with basic epidemiological criteria, 30% of the putative Sma I clusters were inconsistent with the epidemiological criteria compared with 17% of the Kpn I clusters. Among the 98 isolates assigned to clusters by Sma I, only 65% gave concordant results with Kpn I. In contrast, among the 81 isolates assigned to clusters by Kpn I, 92% gave concordant results with Sma I. Finally, clusters that were epidemiologically related to ingestion of raw milk and speci®c water sources correlated better with the typing results based on Kpn I than Sma I. Thus, Kpn I is the enzyme of choice for molecular epidemiology studies of C. jejuni. The combination of continuous epidemiological surveillance and molecular strain typing may be useful for identifying new sources and mechanisms of transmission for community-acquired C. jejuni infection and ultimately for developing new approaches to prevention.
Journal of clinical microbiology, 2000
Six methods for subtyping of Campylobacter jejuni were compared and evaluated with a collection of 90 isolates from poultry, cattle, and sporadic human clinical cases as well as from a waterborne outbreak. The applied methods were Penner heat-stable serotyping; automated ribotyping (RiboPrinting); random amplified polymorphic DNA typing (RAPD); pulsed-field gel electrophoresis (PFGE); restriction fragment length polymorphisms of the flagellin gene, flaA (fla-RFLP); and denaturing gradient gel electrophoresis of flaA (fla-DGGE). The methods were evaluated and compared on the basis of their abilities to identify isolates from one outbreak and discriminate between unrelated isolates and the agreement between methods in identifying clonal lines. All methods identified the outbreak strain. For a collection of 80 supposedly unrelated isolates, RAPD and PFGE were the most discriminatory methods, followed by fla-RFLP and RiboPrinting. fla-DGGE and serotyping were the least discriminative. A...
Journal of clinical microbiology, 2000
Amplified-fragment length polymorphism (AFLP) analysis with the endonucleases BglII and MfeI was used to genotype 91 Campylobacter jejuni subsp. jejuni strains from outbreaks and sporadic cases. AFLP-generated fragments were labeled with fluorescent dye and separated by capillary electrophoresis. The software packages GeneScan and GelCompar II were used to calculate AFLP pattern similarities and to investigate phylogenetic relationships among the genotyped strains. The AFLP method was compared with two additional DNA-based typing methods, pulsed-field gel electrophoresis (PFGE) using SmaI and restriction fragment length polymorphism analysis on PCR products (PCR-RFLP) of the flaA and flaB genes. We found that AFLP analysis of C. jejuni strains is a rapid method that offers better discriminatory power than do both PFGE and PCR-RFLP. AFLP and, to a lesser extent, PCR-RFLP could differentiate strains within the same PFGE profiles, which also makes PCR-RFLP an alternative to PFGE. We we...
BMC microbiology, 2012
Background: Campylobacter jejuni, the most leading cause for bacterial gastroenteritis worldwide, shows a high genetic diversity among its isolates. Recently, we demonstrated the existence of six C. jejuni-groups by combining MLST with six genetic markers. These groups were further characterized by the detection of cj1321-cj1326, fucP, cj0178, cj0755/cfrA, ceuE, pldA, cstII, and cstIII in order (I.) to show further associations between these different genetic markers and MLST CCs. Moreover, different studies were able to associate several of these markers: a sialylated lipoologosaccharide (cstII/III + ), the gamma-glytamyl-transpeptidase (ggt + ), and the absence of a certain allele of the enterochelin-uptake-binding-protein (ceuE 11168 -) with severe campylobacteriosis, bloody diarrhea and unpleasant outcome. Additionally more than half of human Campylobacter-isolates were assigned to a non-livestock clade associated with the absence of cj1321-cj1326. These isolates were considered as mere colonizers. From the combination of marker genes, the ratio of human isolates in a specific group, and clinical data (II.) it should be demonstrated to which of the previous defined groups these Campylobacter-subpopulations, associated with higher virulence, correspond. Results: Besides the marker gene pldA, all new estimated genetic markers show significant differences in their distribution among the various MLST-based groups. Especially the genes for cj1321-cj1326, fucP, cj0178, cj0755/cfrA are widely associated with each other and split the study population into two major and seven intermediate groups substantiating the previous group-definition, whereas cstII and cstIII indicate at least three groups following an independent distribution pattern. Conclusions: Based on these data a group of C. jejuni-isolates characterized by the presence of ansB, dmsA, ggt, and the absence of cj1365c, cj1585c, cj1321-cj1326, fucP, cj0178, cj0755/cfrA, and cstII/III was associated with a higher prevalence in human campylobacteriosis, bloody diarrhea as well as hospitalization and bears obviously a higher virulence for humans. In contrast to that better livestock-adapted groups characterized by the ability to utilize L-fucose and the presence of all of the five identified putative C. jejuni iron-uptake systems as well as cj1321-cj1326, cj1365c, cj1585c, and cstII and/or cstIII (sialylated lipoologosaccharide) is more prevalent in animal hosts and was secondary associated with less severe campylobacteriosis.