Performance of a 70-mer oligonucleotide microarray for genotyping of Campylobacter jejuni
- Sandra Rodin1,
- Anders F Andersson†1, 2,
- Valtteri Wirta†1,
- Lena Eriksson1,
- Marianne Ljungström1,
- Britta Björkholm3,
- Hans Lindmark4 and
- Lars Engstrand1, 3Email author
© Rodin et al; licensee BioMed Central Ltd. 2008
Received: 31 October 2007
Accepted: 08 May 2008
Published: 08 May 2008
Campylobacter jejuni is widespread in the environment and is the major cause of bacterial gastroenteritis in humans. In the present study we use microarray-based comparative genomic hybridizations (CGH), pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) to analyze closely related C. jejuni isolates from chicken and human infection.
With the exception of one isolate, the microarray data clusters the isolates according to the five groups determined by PFGE. In contrast, MLST defines only three genotypes among the isolates, indicating a lower resolution. All methods show that there is no inherit difference between isolates infecting humans and chicken, suggesting a common underlying population of C. jejuni. We further identify regions that frequently differ between isolates, including both previously described and novel regions. Finally, we show that genes that belong to certain functional groups differ between isolates more often than expected by chance.
In this study we demonstrated the utility of 70-mer oligonucleotide microarrays for genotyping of Campylobacter jejuni isolates, with resolution outperforming MLST.
Campylobacter jejuni is a major cause of human bacterial gastroenteritis in industrialized countries . Infection commonly results in self-limiting gastroenteritis but sequelae may occur, for instance in the form of the Guillain-Barré syndrome causing peripheral neuropathy . The genus Campylobacter is widespread in the environment and constitutes part of the normal flora of birds, cattle and swine. Although there are gaps in our knowledge of the sources of infection, the handling and consumption of chicken meat are considered important routes of transmission [3, 4].
Cases of campylobacteriosis are mainly sporadic but outbreaks do occur, predominantly through contaminated milk and untreated water . Due to the sporadic nature of campylobacter infections, it has proven hard to discern the epidemiological characteristics of the disease. Robust and reproducible typing methods are needed to this end, and a multitude of genotypic methods are now complementing serotyping and other traditional phenotypic methods (for a review, see ). Among these, pulsed-field gel electrophoresis (PFGE) possess high discriminatory power and is widely used for studies of strain relatedness [7–11]. However, PFGE requires strict adherence to standardized protocols, and produces data in the form of band patterns of restriction endonuclease digested fragments which are not readily compared between laboratories. Errors or ambiguities in the assignment of bands may also occur [12, 13].
A multilocus sequence typing (MLST) scheme assesses genetic differences by nucleotide sequence determination of approximately 500 bases in each of seven loci . The strain discriminatory performance is highly dependent on the screened loci, which are selected to represent slowly evolving genes under stabilizing selection pressure, supposedly unaffected by antigen variation or genomic rearrangements. Each allele is assigned a number based on sequences in the MLST database . Thus, each isolate is described by a seven-digit sequence type (ST), which is further grouped according to lineage into clonal complexes, defined as groups of isolates with identical alleles at ≥4 loci. The MLST scheme has been used in studies of the population structure of clinical and veterinary isolates of C. jejuni [10, 16–18]. The discriminatory power was comparable to that of multilocus enzyme electrophoresis , and amplified fragment length polymorphism , but did not reach that of PFGE in a study of epidemiologically related isolates .
Comparative genomic hybridizations (CGH) using genome-wide DNA microarrays have proven useful in studies of intraspecies diversity for a number of bacterial species [20–23]. Determination of the full genome sequence of C. jejuni strain NCTC 11168  allowed construction of microarrays for studies of the genetic relationship between campylobacter. Using strain NCTC 11168 as reference, several studies have demonstrated a high degree of intraspecies variability concentrated to defined genomic regions, particularly affecting loci coding for lipooligosaccharides, flagellar modification, and DNA restriction-modification systems [25–31]. CGH may also elucidate sources of infection, transmission routes and virulence of bacteria [31, 32].
Few studies have exploited the power of CGH to evaluate the accuracy and resolution of present genotyping technologies. In the current study we used a whole-genome microarray to study C. jejuni isolates typed with PFGE. We studied closely related pairs of chicken and human isolates, which clustered together in the PFGE analysis, with the aim to dissect the true genetic relationship within and between the pairs. The CGH data in this study were generated using an oligonucleotide array, which was evaluated for its ability to discriminate between present and absent or divergent genes. The results were further compared with MLST results to evaluate the genotyping resolution of the different methods.
Multilocus sequence typing
Summary of the RM1221 vs. NCTC 11168 comparison and the effect of probe-target similarity on the M-values
Number of probes
90 – 93
< = 89
The isolates included in the study showed large differences in the number of probes (range from 30 to 420) that were sequence divergent compared to the reference strain. In total 29% (439/1,527) and 17% (253/1,527) of the probes were variable in more than two and three of the studied isolates, respectively. These numbers are in line with previous CGH studies [25–31]. The variable probes represented genes distributed over the entire chromosome, and showed some local clustering (Figure 2C). Probes exhibiting M-values of >0.75 were classified as representing genes with higher copy numbers in the test isolate compared to the reference strain NCTC 11168. Genes with higher copy numbers were detected in five of the test isolates (three genes in C12, one in C36, nine in H467, six in C20, and two in H312). In isolates C12 and C20 two consecutive genes with higher copy numbers could be detected (C12: Cj0078c and Cj0079c, C20: Cj0967 and Cj0968, and Cj1419c and Cj1420c). The M-values for all probes are available through the ArrayExpress data repository (accession number E-TABM-460).
Clustering and correlation of the different typing methods
Identification of variable regions
Previous CGH studies of C. jejuni have identified 18 genomic regions enriched for genes with diverging sequences [27, 29]. We analyzed the presence of variable genes in the five groups of isolates (A, B, C, D and H) defined by PFGE and microarray clustering. A region was confirmed variable if the calculated average M-value of the group was <-0.75 for at least one of the probes in the region. Using this approach, we noted that all of the previously identified variable regions differed in at least one of the five isolate groups, and further that four variable regions (regions 1, 9, 12 and 13) showed divergence in all five groups (Figure 4B). Also region 11 was highly variable, with four isolate groups showing variability in the region. We further mined our data for additional regions that showed variability in multiple isolates. We found three additional regions, spanning genes Cj0137–Cj0145 (region 19), Cj0356c-Cj0360 (region 20) and Cj1047c-Cj1069 (region 21).
Identification of COG groups enriched for variable regions
We further used the Clusters of Orthologous Groups of proteins (COG) database  to analyze the functional group assignments of the variable genes (M-values of <-0.75 for the corresponding probes). In all isolates genes from multiple COG categories were found variable, indicating that sequence divergence is not restricted to genes encoding specific functions (Figure 4C). Furthermore, using Fisher exact test we identified a significant overrepresentation of divergent genes in the COG category M (cell wall/membrane/envelope biogenesis) and V (defense mechanisms) in several of the isolates (Figure 4D). A strong enrichment of the same categories was observed when the analysis was restricted to genes with high sequence divergence (i.e., M-values of <-2.0). However, when the analysis was carried out using genes with moderate sequence divergence (M-values between -2.0 and -0.75), no significant enrichment of COG categories could be observed. These results suggest that the moderate sequence divergence reflects normal interstrain variability unlikely to affect protein function in any substantial way. Furthermore, the probes with moderate sequence divergence seem to be distributed over the entire length of the chromosome, while the probes with high sequence divergence seem to be more tightly clustered (Figure 2C).
In this study we evaluated three different methods for analysis of the genomic content of closely-related C. jejuni isolates from chicken and humans. The methods tested were CGH using oligonucleotide microarrays, and genotyping by PFGE and by MLST. We first analyzed six pairs of human and chicken isolates which were clustered based on PFGE of KpnI digests. Cluster analysis based on CGH data yielded an identical grouping, with the exception of one isolate. Thus PFGE, which may appear a relatively crude method, produced a phylogenetic tree which coincided well with the one produced by genomic probing through CGH. The data further suggests that there are no genetic markers distinguishing the human from the chicken isolates included in this study. The isolates were subtyped using a previously described MLST scheme . MLST defined three genotypes among the twelve isolates, compared to five defined by PFGE. All eight isolates with PFGE genotypes A (n = 4), B (2) and C (2) were found to belong to sequence type 21. The ST-21 complex has previously been shown to be abundant among isolates from a wide variety of sources [14, 16–19]. The two PFGE genotype D isolates were assigned to ST-48, a sequence type differing from ST-21 in three of the seven loci. Thus, our results suggest that a combined approach using MLST in combination with a second method is necessary to reach a sufficient discriminatory power, at least for resolving epidemiological relationships on a shorter time scale. This conclusion is supported by previous studies [11, 34].
Using the microarray data, we have shown that several previously identified regions [27, 29] are also divergent in isolates investigated in this study. These include regions that are known to be important modulators of the surface-exposed antigenic proteins (e.g., contain genes encoding flagella proteins). In the present study we identified additional regions that are divergent between the isolates, which suggests that additional genome-wide studies are required to fully characterize the variability of the C. jejuni genome. A functional analysis of the variable genes showed that modulation of the surface exposed structures is important for creating variability in the C. jejuni isolates, possibly providing means for avoidance of the host's immune system.
As far as we are aware, this is the first study where a genome-wide oligonucleotide array is used for CGH-based genotyping of C. jejuni. Previous studies have used microarrays based on polymerase chain reaction-amplified probes for analysis of different C. jejuni isolates [25–31]. The main advantage associated with the use of oligonucleotide arrays is the avoidance of extensive cross-hybridization with other regions of the genome and an improved specificity and resolution, allowing detection of smaller differences between the isolates. Also, the design of the probes can be carried out to ensure approximately equal optimal hybridization conditions, avoiding sequence-specific bias in the hybridization signals. However, there are limitations with the oligonucleotide-based CGH platform. The array probes are targeted towards coding regions of the C. jejuni genome, which does not allow for detection of divergence in intergenic regions. Although more specific, the oligonucleotide probes do not allow for detection of single-base changes and lack the possibility to detect short deletions and changes in gene synteny. Also, sequence divergence affecting a non-targeted region of a gene will remain undetected using the oligonucleotide probe approach, suggesting that the true differences between the isolates may be even stronger than reported here. As with all microarrays, the analysis is limited to genes represented on the microarray, in this case genes present in strain NCTC 11168. The design could be improved by adding probes representing genes from other sequenced C. jejuni genomes.
In this study we have investigated the variability of closely related C. jejuni isolates. The comparative genomics hybridization data did not affect the PFGE-based clustering, with the exeption of one isolate which was removed from the fork containing the remaining isolates of the same PFGE type. Nor did we identify any markers predictive of source (human or chicken). We have further shown that MLST-based genotyping needs to be complemented with other methods to achieve similar resolution as is obtained with the other genotyping approaches. We have also demonstrated that extensive variability between isolates is not restricted to the previously identified regions. Finally, certain functional groups (COG groups M and V) show significant enrichment among the variable genes. Collectively, these results demonstrate the importance of unbiased, genome-wide approaches in analysis of differences between isolates of C. jejuni. This will facilitate our future understanding of parameters governing the pathogenic potential of various isolates and allow the design of relevant tools for assessing the genetic diversity and epidemiology of C. jejuni.
Campylobacter isolates and extraction of genomic DNA
Campylobacter spp. isolates (n = 90) were collected from all reported cases of domestically acquired campylobacteriosis in four Swedish regions within the scope of a study conducted in July through October 2003. During the same time period and in the same geographical areas, fresh poultry products from retail were purchased and analyzed for campylobacter. Isolates from both patients and poultry were species identified by polymerase chain reaction  and the C. jejuni isolates subtyped using PFGE and the restriction enzyme SmaI as earlier described . Isolates sharing PFGE genotype with at least one other isolate were further PFGE-genotyped using the restriction enzyme KpnI. Each unique banding pattern was assigned an identifying letter. For the current study, six pairs of C. jejuni isolates with identical SmaI and KpnI genotypes were selected to represent each of the KpnI genotypes A (two pairs), B, C, D and H (Figure 1). Each pair consisted of one chicken and one human isolate originating from the same geographical region. The two completely sequenced strains NCTC 11168  and RM1221  were also included.
All isolates were cultured on blood agar plates at 37°C for 48 h in a microaerophilic environment. Genomic DNA for MLST and microarray analyses was extracted using the DNeasy tissue kit (Qiagen, Hilden, Germany).
Multilocus sequence typing
The seven loci used in the MLST  were polymerase chain reaction amplified using primers and conditions according to the C. jejuni MLST website http://pubmlst.org/campylobacter. Nucleotide sequences were obtained by sequencing of both strands using the BigDye Terminator v3.1 Cycle Sequencing kit and a 3130xl Genetic Analyzer (Applied Biosystems, Foster City, USA). Sequences were assigned allele numbers, and the sequence type (ST) and lineage (clonal complex) of each isolate was determined by interrogating the MLST database.
Comparative genomic hybridizations
Campylobacter jejuni AROS v1.0 oligonucleotide probe set was purchased from Operon Biotechnologies (Cologne, Germany). The set consisted of 70-mer oligonucleotides representing 1,546 open reading frames (ORFs) from strain NCTC 11168, 51 ORFs from the virulence plasmid pVir from strain 81–176, and 4 ORFs from plasmid pCJ01 from strain 21190. The oligonucleotides were printed in triplicates on CodeLink Activated slides (GE Healthcare, Uppsala, Sweden) and processed according to the manufacturer's instructions. For probe annotation the version 1.3.2 (dated May 16, 2007) of the OMAD database  was used. Additional details on the microarray production are available through the ArrayExpress microarray data repository (accession number A-MEXP-925) .
Test DNA extracted from the twelve C. jejuni isolates was co-hybridized with reference DNA extracted from strain NCTC 11168 in one hybridization per isolate. Additional hybridizations were performed to compare strains NCTC 11168 and RM1221, and to compare two separate target preparations (culturing, DNA extraction, labeling and hybridization) of the NCTC 11168 strain to establish the magnitude of technical noise in the experimental setup. Both sets of additional hybridizations were carried out in a dye-swap manner.
For labeling, 3 μg genomic DNA in 21 μL water was sonicated for 30 s. Fluorescent Cy3 or Cy5 dyes were incorporated in a mixture of 15 μg random octamers, 40 U of Klenow enzyme (Invitrogen, Paisley, UK), 6 nmol of each dATP, dGTP and dCTP, 3 nmol dUTP, and 50 nmol Cy3-/Cy5-dUTP (GE Healthcare, Uppsala, Sweden) in 1 mM Tris pH 8.0, 100 μM EDTA. The mixture was incubated at 37°C for 2 h after which 5 μL 0.5 M EDTA was added. Unincorporated dye was removed using the Microcon-30 columns (Millipore AB, Solna, Sweden) and the dye incorporation efficiency measured using a Nanodrop ND-1000 spectrophotometer (Nanodrop, Rockland, USA). Reference DNA was combined with an equal amount of reciprocally labeled test DNA, dried down using a speed vac, resuspended in 100 μL hybridization buffer (5 × SSC, 50% formamide, 0.1% SDS, 0.1 μg/μL tRNA), and denatured for 2 min at 95°C. The samples were cooled on ice, transferred to the microarrays and hybridized for 16 h at 42°C. The arrays were then washed once for 5 min with 2 × SSC, 0.1% SDS at 42°C, once for 5 min with 0.1 × SSC, 0.1% SDS at room temperature, and four times for one minute with 0.1 × SSC at room temperature. All washing steps were done under agitation. The slides were dried by brief centrifugation at low speed.
The microarrays were scanned using a GenePix 4000B scanner (Molecular Devices, Sunnyvale, USA). Features were identified and fluorescence intensities extracted using the irregular feature-finding approach implemented in GenePix Pro 5.1 (Molecular Devices). Further analysis was carried out in the R environment for statistical computing  using the aroma , Bioconductor  and kth-packages . No subtraction of the local background was carried out, as this was found to slightly increase the variability between replicated features. A feature was considered unreliable and removed if: a) the feature contained less than 55 pixels, or if for both channels b) 10% or more of the pixels were below the signal intensity of the local background plus two standard deviations of the background, c) the signal-to-noise ratio was below 3, d) the signal was saturated, or e) the intensity was below the mean signal of negative controls (probes with random sequence). The design of the probe set is based on the genome sequence of the NCTC 11168 strain, and hence absent or sequence divergent genes in the test isolate (labeled in Cy5) compared to the reference strain (Cy3) show negative log2 (Cy5/Cy3) ratio values (M-values). Data normalization was carried out in a block-wise manner assuming equal sums of the two channels using a non-divergent set of probes. These probes were obtained after removal of 20% of the probes with the most negative M-values. After normalization, replicates of each probe were averaged, discarding probes that had only one available measurement. The microarray dataset is available through ArrayExpress (accession number E-TABM-460) .
We thank A. Waldén and P. Nilsson at the Swedish Royal Institute of Technology for printing the microarrays. Strain RM1221 was a kind gift from R. E. Mandrell, United States Department of Agriculture, USA.
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