Application of single-strand conformation polymorphism and denaturing gradient gel electrophoresis for fla sequence typing of Campylobacter jejuni

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 3' end of the flaA to the 3' end 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.     

High-resolution genotyping of Campylobacter strains isolated from poultry and humans with amplified fragment length polymorphism fingerprinting.

For epidemiological studies of Campylobacter infections, molecular typing methods that can differentiate campylobacters at the strain level are needed. In this study we used a recently developed genotyping method, amplified fragment length polymorphism (AFLP), which is based on selective amplification of restriction fragments of chromosomal DNA, for genetic typing of Campylobacter jejuni and Campylobacter coli strains derived from humans and poultry. We developed an automated AFLP fingerprinting method in which restriction endonucleases HindIII and HhaI were used in combination with one set of selective PCR primers. This method resulted in evenly distributed band patterns for amplified fragments ranging from 50 to 500 bp long. The discriminatory power of AFLP was assessed with a C. jejuni strain, an isogenic flagellin mutant, and distinct C. jejuni strains having known pulsed-field gel electrophoresis and fla PCR-restriction fragment length polymorphism genotypes. Unrelated C. jejuni strains produced heterogeneous patterns, whereas genetically related strains produced similar AFLP patterns. Twenty-five Campylobacter strains obtained from poultry farms in The Netherlands grouped in three C. jejuni clusters that were separate from a C. coli cluster. The band patterns of 10 C. jejuni strains isolated from humans were heterogeneous, and most of these strains grouped with poultry strains. Our results show that AFLP analysis can distinguish genetically unrelated strains from genetically related strains of Campylobacter species. However, desirable genetically related strains can be differentiated by using other genotyping methods. We concluded that automated AFLP analysis is an attractive tool which can be used as a primary method for subtyping large numbers of Campylobacter strains and is extremely useful for epidemiological investigations.     

Fingerprinting of Campylobacter jejuni by using resolution-optimized binary gene targets derived from comparative genome hybridization studies

The aim of this investigation was to exploit the vast comparative data generated by comparative genome hybridization (CGH) studies of Campylobacter jejuni in developing a genotyping method. We examined genes in C. jejuni that exhibit binary status (present or absent between strains) within known plasticity regions, in order to identify a minimal subset of gene targets that provide high-resolution genetic fingerprints. Using CGH data from three studies as input, binary gene sets were identified with "Minimum SNPs" software. "Minimum SNPs" selects for the minimum number of targets required to obtain a predefined resolution, based on Simpson's index of diversity (D). After implementation of stringent criteria for gene presence/absence, eight binary genes were found that provided 100% resolution (D=1) of 20 C. jejuni strains. A real-time PCR assay was developed and tested on 181 C. jejuni and Campylobacter coli isolates, a subset of which have previously been characterized by multilocus sequence typing, flaA short variable region sequencing, and pulsed-field gel electrophoresis. In addition to the binary gene real-time PCR assay, we refined the seven-member single nucleotide polymorphism (SNP) real-time PCR assay previously described for C. jejuni and C. coli. By normalizing the SNP assay with the respective C. jejuni and C. coli ubiquitous genes, mapA and ceuE, the polymorphisms at each SNP could be determined without separate reactions for every polymorphism. We have developed and refined a rapid, highly discriminatory genotyping method for C. jejuni and C. coli that uses generic technology and is amenable to high-throughput analyses.     

The occurrence of Campylobacter subtypes in environmental reservoirs and potential transmission routes

AIM: To identify potential reservoirs and transmission routes of human pathogenic Campylobacter spp. METHODS AND RESULTS: An enrichment PCR method for the detection and identification of Campylobacter jejuni and/or Campylobacter coli in faecal, food and river water samples was applied to 1450 samples of 12 matrix types obtained from a defined geographical area. PCR-positive samples were cultured to yield isolates for typing, and the data for 616 C. jejuni isolates obtained. Serotyping and SmaI macrorestriction profiling using pulsed field gel electrophoresis revealed a high level of diversity within the isolates from each matrix. Campylobacter jejuni and C. coli subtypes indistinguishable from those obtained from human cases were detected in most of the matrices examined. No Campylobacter isolates were isolated from possum faeces. CONCLUSIONS: Ten of the 12 matrices examined may be involved in the transmission of human campylobacteriosis as they contained Campylobacter subtypes also isolated from clinical cases. SIGNIFICANCE AND IMPACT OF THE STUDY: Results indicate that, for this rural population, a range of potential transmission routes that could lead to campylobacteriosis exist. Their relative importance needs to be assessed from an exposure assessment standpoint.     

Genotypic Diversity among Campylobacter jejuni Isolates in a Commercial Broiler Flock

Analysis of nucleic acid polymorphism in the flagellin genes of Campylobacter jejuni was used to investigate genetic diversity among Campylobacter spp. in a commercial broiler flock. Three hundred single colonies of C. jejuni were isolated from fecal samples collected weekly for 3 weeks immediately before slaughter. Both the flaA and flaB genes were amplified by PCR, and the PCR product was digested with the restriction enzyme AluI. The fragments generated were then analyzed by agarose gel electrophoresis. Among the 300 recovered isolates, five different restriction fragment length polymorphism profiles were observed. Three of these profiles were dominant during the course of the study, and the other two profiles were detected at low frequency. Analysis of genetic variation in C. jejuni over the course of an experimental infection lasting 7 weeks indicated that there was no obvious drift in the flagellin gene type. These findings demonstrate that a range of bacterial genotypes can constitute the bacterial population within a commercial poultry flock, with the most likely sources of these types being multiple environmental exposure and/or genetic drift within the population. This degree of diversity must be considered in epidemiological analyses which utilize genetic typing methods that investigate Campylobacter contamination of any food source, including poultry, to ensure that the total gene pool for C. jejuni is evaluated.     

Comparison of Campylobacter jejuni PFGE and Penner subtypes in human infections and in water samples from the Taieri River catchment of New Zealand

AIMS: To determine the degree of overlap in strain types of Campylobacter jejuni isolated from clinical cases and water samples from the Taieri catchment in the South Island of New Zealand. METHODS AND RESULTS: Thermophilic Campylobacter were collected from human cases of infection, the main stem of the Taieri River and streams within distinct land-use types over a 1-year period. Campylobacter jejuni (187 isolates) and Campylobacter lari (four isolates) were identified using a multiplex polymerase chain reaction protocol. Isolates were typed by the Penner method and pulsed-field gel electrophoresis (PFGE) utilizing two restriction endonucleases. Several serotypes and PFGE types occurred in both water samples and clinical cases when the restriction profiles for each enzyme were considered separately. However, when PFGE profiles and serotyping were combined, there was no overlap between Camp. jejuni types from water and clinical cases. CONCLUSIONS: The results of this study indicate that recreational water in the Taieri catchment is not a major source of campylobacteriosis in the Dunedin area. SIGNIFICANCE AND IMPACT OF THE STUDY: This study suggests the risk of acquiring campylobacteriosis from surface waters in the Taieri catchment is considerably lower than previously predicted and highlights the necessity of using two endonucleases in PFGE typing.     

Heterogeneity of non-serotypable Campylobacter jejuni isolates

Several phenotypic and genotypic methods are currently used to identify subtypes of Campylobacter jejuni isolates. Of the phenotypic methods one, i.e. serotyping based on the heat stable antigen, is often hindered by the relatively large number of un-typable (NT) strains. Little is known, however, about the heterogeneity of the group formed by these NT strains. Therefore we serotyped 92 Hungarian, non-outbreak C. jejuni isolates and subjected the 28 NT strains to molecular analysis using PCR-RFLP of the flaA gene and to pulsed-field gel electrophoresis. With both methods the NT strains were classified into several molecular types (17 and 25, respectively), while the number of subgroups based on the results of the two techniques combined was twenty-six. These results indicate that the NT group of strains is extremely heterogeneous in Hungary, and the epidemiological connection between two NT isolates cannot be established or excluded without the use of molecular typing techniques.     

Longitudinal study of Campylobacter jejuni bacteriophages and their hosts from broiler chickens

A longitudinal study of bacteriophages and their hosts was carried out at a broiler house that had been identified as having a population of Campylobacter-specific bacteriophages. Cloacal and excreta samples were collected from three successive broiler flocks reared in the same barn. Campylobacter jejuni was isolated from each flock, whereas bacteriophages could be isolated from flocks 1 and 2 but were not isolated from flock 3. The bacteriophages isolated from flocks 1 and 2 were closely related to each other in terms of host range, morphology, genome size, and genetic content. All Campylobacter isolates from flock 1 were genotypically indistinguishable by pulsed-field gel electrophoresis (PFGE). PFGE and multilocus sequence typing indicated that this C. jejuni type was maintained from flock 1 to flock 2 but was largely superseded by three genetically distinct C. jejuni types insensitive to the resident bacteriophages. All isolates from the third batch of birds were insensitive to bacteriophages and genotypically distinct. These results are significant because this is the first study of an environmental population of C. jejuni bacteriophages and their influence on the Campylobacter populations of broiler house chickens. The role of developing bacteriophage resistance was investigated as this is a possible obstacle to the use of bacteriophage therapy to reduce the numbers of campylobacters in chickens. In this broiler house succession was largely due to incursion of new genotypes rather than to de novo development of resistance.     

Subtyping of Campylobacter jejuni Penner serotypes 9, 38 and 63 from human infections, animals and water by pulsed field gel electrophoresis and flagellin gene analysis

Pulsed field gel electrophoresis and PCR-RFLP flagellin gene profiling were used to discriminate 44 isolates of Campylobacter jejuni Penner heat stable (HS) serotypes 9, 38 and 63 from sporadic human infections and other sources. Genomic similarities between HS9 and HS38 strains were demonstrated. HS63 and HS1 strains of Camp. jejuni ssp. jejuni were similar but were genomically distinct from Camp. jejuni ssp. doylei HS63. The molecular analyses provided a basis for assessing associations between cross-agglutinating strains of Camp. jejuni and for subtyping within those serogroups.     

Detection and investigation of Campylobacter jejuni by polymerase chain reaction-restriction fragment length polymorphism analysis

A molecular typing approach for Campylobacter jejuni with restriction fragment length polymorphism (RFLP) analysis of the flagellin gene flaA in C. jejuni, was generated and studied. Using polymerase chain reaction (PCR)-RFLP with the restriction endonuclease Mbo I, it was demonstrated that C. jejuni could be divided into four types. Genotypic analysis of C. jejuni by PCR-RFLP is a valuable technique for epidemiological typing.     

Evidence that certain clones of Campylobacter jejuni persist during successive broiler flock rotations

Through the national surveillance program for Campylobacter spp., nine broiler chicken farms that were infected with Campylobacter jejuni in at least five rotations in 1998 were identified. One additional farm, located at the island of Bornholm where divided slaughter is used extensively, was also selected. Twelve broiler houses located on 10 farms were included in the study. The C. jejuni isolates collected from the selected houses during the surveillance were typed using fla typing and macrorestriction profiling (MRP), and a subset of the isolates, representing each of the identified clones, was serotyped according to the Penner scheme. Pulsed-field gel electrophoresis typing using SmaI and KpnI revealed that the majority of houses (11 of 12) carried identical isolates in two or more broiler flocks. Such persistent clones were found in 63% of all flocks (47 of 75). The majority of persistent clones (7 of 13) had fla type 1/1, but MRPs distinguished between isolates from different houses, and fla type 1/1 clones belonged to different serotypes. Seven houses carried persistent clones that covered an interval of at least four broiler flock rotations, or at least one half year. The dominant fla type (1/1) was represented by 44% of isolates, or by at least one isolate from 31 of 62 broiler flocks. This significantly exceeded the prevalence of fla type 1/1 C. jejuni isolates that we have estimated from other studies and suggests that isolates carrying this fla type are overrepresented in flocks with recurrent Campylobacter problems. The MRPs of clones belonging to fla type 1/1 serotype O:2 isolated from persistently infected flocks shared a high percentage of bands compared to the remaining isolates, indicating that some clones that have the ability to cause persistent infections in broiler farms are highly related to each other.     

Location of epitopes on Campylobacter jejuni flagella.

Flagella were isolated from strains of Campylobacter jejuni belonging to different heat-labile serogroups and from a strain of Campylobacter fetus, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the flagellin molecular weights (Mr) were approximately 62,000. The flagellins were cleaved by hydrolysis with cyanogen bromide, and sodium dodecyl sulfate-urea peptide gel electrophoresis showed that the C. jejuni flagellins were structurally similar, and differed from C. fetus flagellin. Immunochemical analysis by Western blotting, enzyme-linked immunosorbent assay, immune electron microscopy, and immunoprecipitation with polyclonal and monoclonal antibodies revealed the presence of both internal and surface-exposed epitopes. The internal epitopes were antigenically cross-reactive and linear, and in the case of C. jejuni flagellin were located on cyanogen bromide peptides of apparent Mr 22,400 and 11,000. Antigenically cross-reactive epitopes were also present on an Mr 43,000 cyanogen bromide peptide of C. fetus flagellin. The Mr 22,400 peptide of C. jejuni VC74 flagellin also carried closely positioned internal linear epitopes for two monoclonal antibodies. One epitope was strain specific, while the other was shared by some but not all Campylobacter flagellins. The flagella of C. jejuni VC74 also displayed both surface-exposed antigenically cross-reactive and surface-exposed serospecific epitopes. Both linear and conformational epitopes contributed to the serospecificity of C. jejuni VC74 flagella, and a linear serospecific epitope was located on a cyanogen bromide peptide of apparent Mr 4,000.     

Genotyping of Campylobacter jejuni strains from Danish broiler chickens by restriction fragment length polymorphism of the LPS gene cluster

AIMS: To apply and evaluate LG (LPS genes) genotyping, which is a genotyping method based on a cluster of genes involved in the synthesis of surface lipopolysaccharides (LPS) in Campylobacter species, for typing of Campylobacter jejuni isolates obtained from Danish broiler chickens. Furthermore, the LG genotyping method was used to study the genetic stability of four C. jejuni strains after gastrointestinal passage through experimentally infected chickens. METHODS AND RESULTS: In the present study, the LG genotyping method was modified with respect to the restriction enzymes used. To validate the method, 63 Penner serotype reference strains and 107 C. jejuni chicken isolates, representing the most common Penner serotypes of C. jejuni in Danish poultry, were selected for typing. The method was successfully used for typing all isolates and the LG genotype profiles were reproducible. There were no changes in the LG genotype of the C. jejuni strains obtained after experimental passage through chickens. CONCLUSIONS: All C. jejuni strains obtained from broiler chickens were typeable by the LG genotyping method. Application of the RsaI restriction enzyme improved the method in terms of ease and consistency of analyses and increase of discriminatory power. SIGNIFICANCE AND IMPACT OF THE STUDY: The LG genotyping method is a valuable tool for typing C. jejuni isolates obtained from poultry. However, the association between Penner serotyping based on passive haemagglutination of heat-stable antigens and LG genotyping was low when applied to poultry isolates. This is in contrast to previous studies on isolates of human origin that reported a high correlation between results obtained by the two typing methods (Shi et al. 2002).     

耐甲氧西林金黄色葡萄球菌分子分型研究进展

近年来,耐甲氧西林金黄色葡萄球菌在全世界各地感染率和分离率不断提高,已成为目前院内感染的重要病原菌之一。运用有效、可靠、廉价的分子分型方法对分析耐甲氧西林金黄色葡萄球菌的流行病学特征及来源,对制定控制院感及流行的措施非常重要的。本研究概述了各种分子分型方法的原理及比较,如SCCmec分型、脉冲场凝胶电泳分型、多位点序列分型、葡萄球菌A蛋白分型和毒力因子分型等。脉冲场凝胶电泳仍然是暴发流行中MRSA分子分型的金标准,而其他分型方法更适合用于检测菌株的变异和建立国际监测。     

Linkage analysis by two-dimensional DNA typing.

In two-dimensional (2-D) DNA typing, genomic DNA fragments are separated, first according to size by electrophoresis in a neutral polyacrylamide gel and second according to sequence by denaturing gradient gel electrophoresis, followed by hybridization analysis using micro- and minisatellite core probes. The 2-D DNA typing method generates a large amount of information on polymorphic loci per gel. Here we demonstrate the potential usefulness of 2-D DNA typing in an empirical linkage study on the red factor in cattle, and we show an example of the 2-D DNA typing analysis of a human pedigree. The power efficiency of 2-D DNA typing in general is compared with that of single-locus typing by simulation. The results indicate that, although 2-D DNA typing is very efficient in generating data on polymorphic loci, its power to detect linkage is lower than single-locus typing, because it is not obvious whether a spot represents the presence of one or two alleles. It is possible to compensate for this lower informativeness by increasing the sample size. Genome scanning by 2-D DNA typing has the potential to be more efficient than current genotyping methods in scoring polymorphic loci. Hence, it could become a method of choice in mapping genetic traits in humans and animals.     

Comparison between agarose gel electrophoresis and capillary electrophoresis for variable numbers of tandem repeat typing of Mycobacterium tuberculosis

Variable numbers of tandem repeat (VNTR) typing of Mycobacterium tuberculosis was performed on 54 strains including 23 strains derived from 9 outbreaks. PCR amplicon sizes of 12 mycobacterial interspersed repetitive unit tandem repeat loci were measured using both agarose gel electrophoresis and capillary electrophoresis. Similarities using agarose gel electrophoresis of Euclidian distances among the 23 strains derived from the 9 outbreaks were significantly lower than that using capillary electrophoresis (Wilcoxon signed ranks test, P < 0.01). By clustering analysis using unweighted pair group method using arithmetic averages, all of the 23 strains derived from the 9 outbreaks were each clustered with more than 90% similarities based on the distance using capillary electrophoresis. In contrast, differential clusters with more than 90% similarity were observed with only 7 strains derived from 3 outbreaks when analyzed by agarose gel electrophoresis. These results indicated that measurement of PCR amplicon size of tandem repeat loci should be carried out using capillary electrophoresis and that agarose gel electrophoresis is not suitable for clustering analysis of M. tuberculosis VNTR typing.     

Inhibition of DNAse activity in PFGE analysis of DNA from Campylobacter jejuni

DNAse-positive strains of Campylobacter jejuni degrade their chromosomal DNA during standard preparative procedures before pulsed-field gel electrophoresis (PFGE). A simple method for inactivation of this DNAse activity is described. Formaldehyde fixation of the bacterial cells resulted in the preservation of the DNA in a state suitable for restriction digestion and subsequent electrophoretic analysis.     

Comparison of multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), and amplified fragment length polymorphism (AFLP) for genetic typing of Staphylococcus aureus

We compared multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), and amplified fragment length polymorphism (AFLP) for typing of Staphylococcus aureus and show that the methods yield similar results, although with differences in resolving power and reproducibility. Epidemiological conditions should determine which is the optimal typing method to be used.     

Relationships between bacterial genotypes and in vitro virulence properties of Campylobacter jejuni and Campylobacter coli isolated from turkeys

AIMS: Campylobacter isolates from turkeys were genotyped and characterized by their in vitro virulence properties. Relationships between bacterial genotypes and virulence properties were analysed. METHODS AND RESULTS: Isolates were analysed by pulsed-field gel electrophoresis and fla typing. The toxin production was determined on the phenotypic level using a CHO-K1 cell culture model and on the genotypic level using PCR for detection of the cdtA, cdtB and cdtC genes. Although the cdtB gene was detected from 100% of the Campylobacter jejuni and Campylobacter coli isolates we observed three different morphological pictures on the cells. Cytotoxicity was associated with cell distension or cell rounding. All four Camp. coli strains and one Camp. jejuni strain did not produce any cytotoxic changes on the cells. Adhesion, invasion and survival of Campylobacter isolates were determined in a Caco-2 cell culture model. All isolates adhered to and invaded Caco-2 cells, whereas 64.7% of the strains survived for 48 h in the cells. CONCLUSION: Seventeen Campylobacter isolates from turkeys were classified into four groups with regard to their in vitro abilities. Jackknife analysis revealed a strong association between these groups and genotype clusters. SIGNIFICANCE AND IMPACT OF THE STUDY: Typing methods have generally failed to identify strains with specific virulence properties. This study suggests that a relationship between subgroups of Campylobacter with common in vitro virulence characteristics and genotypes exist.     

Prevalence and genetic diversity of Campylobacter spp. in environmental water samples from a 100-square-kilometer predominantly dairy farming area

Water samples were taken systematically from a 100-km2 area of mainly dairy farmland in northwestern England and examined for Campylobacter spp. Pulsed-field gel electrophoresis-restriction fragment length polymorphism (PFGE-RFLP) and flaA strain typing of Campylobacter jejuni and Campylobacter coli isolates were done. Data on the water source and the adjacent environment were recorded and examined as explanatory variables. Campylobacter spp. were isolated from 40.5% (n = 119) of the water samples tested. C. jejuni was isolated from 14.3%, C. coli was isolated from 18.5%, and Campylobacter lari was isolated from 4.2% of the samples. Campylobacter hyointestinalis was not isolated from any water source. The difference in prevalence between water types (trough, running, and standing) was significant (P = 0.001). C. jejuni was the species most commonly isolated from trough-water and running-water sources, while C. coli was the most frequently isolated from standing water (P < 0.001). No association was found between the presence of Escherichia coli and that of Campylobacter spp. The final multivariable logistic regression model for Campylobacter spp. included the following variables: water source, soil type, aspect, and amount of cattle fecal material in the environment (fecal pat count). Strain typing demonstrated a diverse population of C. jejuni and the presence of a common C. coli flaA type that was widely distributed throughout the area. Most of the isolates within the common flaA type were discriminated by PFGE-RFLP. These findings suggest a possible role for environmental water in the epidemiology of Campylobacter spp. in a farming environment.