A new multiplex PCR and sequencing approach is presented as a modular, three-level genetic characterization system for C. jejuni and C. coli. This approach covers general identification, typing, and determination of antibiotic resistance. Previously established 16S rRNA and rpoB gene sequencing was applied for clear-cut identification of isolates. The MLST scheme was optimized to comprise a single and universal primer set for both species. Optimized primers were also designed for typing based on flaA and flaB genes, which can be included as an additional method and can be used alone or in combination with MLST. Finally, determination of macrolide and quinolone resistances was achieved by 23S rRNA and gyrA gene sequencing, respectively.
In order to minimize and optimize both handling and reagents, a multiplex PCR was set up, combining targets that differ in size, so that they yield specific products that are all amplified with equal efficiency. This resulted in the optimal combination of 12 targets in three AGs, thereby achieving a fourfold reduction in the number of PCRs and an optional single PCR for the 16S rRNA gene. The number of targets and resulting AGs described are exhaustive, and certainly not all 13 targets will be used for genetic characterization. Moreover, interests of laboratories in the various modules might be different. The reason for including so many targets was both to show proof of principle for the multiplex approach and to assess the usefulness of the individual modules. While the method was being evaluated, several target genes were combined, and the most promising have been chosen for the study of the C. jejuni and C. coli strain set collected in Switzerland.
In our experience, a purification step for PCR products is necessary to obtain high-quality sequences, which cannot be achieved if residual primers and other components of the PCR remain during the sequencing reaction. Column purification is normally used, but this method is expensive and inconvenient for high numbers of samples. An enzymatic purification step proved highly suitable and resulted in a significant improvement in sequence quality compared to nonpurified sample results, thus becoming a prerequisite for easy and efficient routine sequence analysis. Previously prepared sequencing plates containing the appropriate primers contributed to optimal handling during the preparation of sequencing reactions, and these plates can be stored at −20°C until they are used and are stable for at least several months. The format can be simply adapted to strips or single tubes, depending on the combination of targets and laboratory needs. Direct purification of sequencing reaction mixtures by a simple single-step ethanol precipitation is possible, and afterwards, the plates, strips, or tubes can be directly loaded on an automated sequencer without further transfer to new tubes.
A large collection of C. jejuni and C. coli strains from various sources were analyzed by the newly developed multiplex approach, which proved highly suitable, especially for MLST. High-quality, unambiguous sequence data could be generated by this procedure. The sequences of the various MLST target genes can be used in the assignment of classical STs after editing, or the full-length sequences can be used for further phylogenetic analysis using the appropriate software. Whereas STs provide easily comparable results for epidemiological purposes, phylogenetic analysis clearly shows the genetic relationships between isolates and thus also allows the separation of the two species. Moreover, while not all of the polymorphic sites located in the additional sequence protruding from the MLST target sequence segments used for typing by PubMLST influence the ST, they might allow further separation of strains belonging to the same ST, thereby increasing the resolution of the method.
The analyzed strain set represented a highly variable group of isolates, which is certainly based on the absence of epidemiological relationship of samples. Nonetheless, this study provides for the first time an overview of the various STs that can be found in Switzerland. The predominant CCs for C. jejuni
were CC21 and CC45, which is the case in other European countries, indicating the wide distribution of these types (4
). Strains representing STs of both CCs were found in human and various animal species, except pigs. For C. coli
, the greatest number of isolates were from ST845, which is found mainly in pigs, but also in poultry, and the newly determined ST3336, which is also isolated in both animal species. Interestingly, there was a relatively high number of STs detected and described for the first time, indicating that specific types are present in Switzerland that have not yet been found in other countries. More systematic studies with defined sample sets would help clarify this situation. Moreover, continuous probing and sampling of potentially contaminated food products, especially chicken, over a defined period of time and comparison of the STs detected with those isolated from human cases would lead to information about the potential risk of infection and provide data for intervention and prevention measures.
The typing of strains based on either flaA
gave nearly overlapping results, which was also reflected in the 98.5% congruence between the two methods. The flaA
gene showed higher discriminatory power than the flaB
gene. However, in combination with MLST, flaB
showed a slightly higher discriminatory ability than flaA
. Moreover, the amplification of flaA
was especially problematic in C. coli
strains when the multiplex approach was used, and only the application of a specially designed optional forward primer for the amplification and sequencing of flaA
solved the problem and resulted in high-quality sequences. However, this solution was not suitable for the multiplex approach. Since flaB
was more stably amplified (99.4% of all strains) and is less prone to recombination than flaA
), the former might be more suitable for typing, especially in combination with MLST.
The fla genes clustered strains in a very different way than MLST, and the isolates were distributed independently of their STs (Fig. and ), reflecting the fact that the two are different typing approaches and cannot be directly compared. This is also indicated by the absence of congruence between the two typing methods. In combination with MLST, the fla genes increased the discriminatory power of the method, which could be helpful in certain situations.
The proper identification of Campylobacter
requires experience and might be difficult. Moreover, Campylobacter
species other than C. jejuni
and C. coli
can be isolated from food poisoning and enteritis patients. In such cases, identification based on genetic markers, e.g., 16S rRNA and rpoB
genes, might be helpful (15
). The resolution of rpoB
was higher than that of the 16S rRNA gene and even allowed separation between C. jejuni
and C. coli
(Fig. ). Therefore, the rpoB
gene might be fully sufficient for the identification of Campylobacter
species, whereas the 16S rRNA gene might be helpful in identifying closely related and sometimes confounded species, such as those from the genus Arcobacter
evolves rather rapidly (31
), and intra- and intergenomic changes not only occur in the environment, but also as a consequence of storage, culture, and passage in vitro. This might result in changes in the nucleotide sequences of different genes and should be taken into consideration when typing strains that have been subcultured over significant amounts of time (9
). We have addressed this question by analyzing sequences of highly passaged strains with their progenitor. We found that the genes used for MLST and fla
typing remained unchanged after more than 200 generations of in vitro subcultivation and are thus well suited for epidemiological investigation, an aspect that has not yet been addressed.
Both gene targets used for the genetic determination of antibiotic resistance to macrolides and quinolones could be efficiently sequenced by the multiplex approach. Moreover, mutations described in the literature as conferring antibiotic resistance were in all cases confirmed by the phenotypic MIC assays. None of the other observed additional mutations were associated with phenotypic resistance. Therefore, the included module for the genetic determination of antibiotic resistance is a highly valuable tool for the analysis of C. jejuni and C. coli. Analysis of isolates collected in Switzerland showed that none of the C. jejuni isolates were resistant to macrolides, whereas almost 21% of C. coli strains showed resistance against this group of antibiotics. With quinolones, 31% of C. jejuni and 40% of C. coli isolates were resistant. Finally, 6% of C. coli strains showed resistance to both classes of antibiotics. This reflects the fact that C. jejuni is predominantly found in poultry, whereas C. coli is mainly isolated from pigs, and antibiotic treatments used with the two animal species are different. The presence of antibiotic resistances demands the prudent use of these antibiotics in animal farming, especially in poultry and pig production.
Interestingly, one human isolate, which was clearly identified as C. coli
, had a quinolone-resistant defining gyrA
gene variant usually found in C. jejuni
strains. This might be the result of recombination between the two species, a phenomenon which they are well known for (31
To improve and facilitate Campylobacter
genotyping, not only on the experimental level, but also on the analytical level, a combined C. jejuni
and C. coli
Web-based IDNS application service has been developed and made available by SmartGene. Analogous to other usages recently described (32
), this platform allows the import of trace files from sequencers, editing, and proofreading by the integrated Proofreader, as well as straight allele, ST, and CC determination over an automated link/submission to the PubMLST database. In order to respond to questions related to epidemiology, the information on strains, their sequences, and final typing results are stored and can be cross-compared. Moreover, to facilitate multicenter collaborations, the software supports online networking between laboratories. While access to this system is protected, the Web technology allows laboratories to be easily connected so that they may access and share their data.
In summary, the MLST scheme for C. jejuni and C. coli was generalized, improved, and automated by establishing a multiplex approach. The approach was successfully applied in its most comprehensive form, including 13 target genes, to more than 300 C. jejuni and C. coli strains, yielding new information on types and antibiotic resistances of strains in Switzerland. Many laboratory-specific adaptations to the format (plates, strips, or tubes), as well as to the actual need (identification, MLST, fla typing, antibiotic resistance status, and their combinations) are possible. An IDNS platform allows easy and straightforward typing of isolates, as well as epidemiological analysis and strain tracing. The described approach contributes to accurate cost- and time-efficient monitoring and tracing of strains and to the development of effective prevention and intervention measures for Campylobacter infection.