Multiplex Strategy for Multilocus Sequence Typing, fla Typing, and Genetic Determination of Antimicrobial Resistance of Campylobacter jejuni and Campylobacter coli Isolates Collected in Switzerland

Abstract

We present an optimized multilocus sequence typing (MLST) scheme with universal primer sets for amplifying and sequencing the seven target genes of Campylobacter jejuni and Campylobacter coli. Typing was expanded by sequence determination of the genes flaA and flaB using optimized primer sets. This approach is compatible with the MLST and flaA schemes used in the PubMLST database and results in an additional typing method using the flaB gene sequence. An identification module based on the 16S rRNA and rpoB genes was included, as well as the genetic determination of macrolide and quinolone resistances based on mutations in the 23S rRNA and gyrA genes. Experimental procedures were simplified by multiplex PCR of the 13 target genes. This comprehensive approach was evaluated with C. jejuni and C. coli isolates collected in Switzerland. MLST of 329 strains resulted in 72 sequence types (STs) among the 186 C. jejuni strains and 39 STs for the 143 C. coli isolates. Fourteen (19%) of the C. jejuni and 20 (51%) of the C. coli STs had not been found previously. In total, 35% of the C. coli strains collected in Switzerland contained mutations conferring antibiotic resistance only to quinolone, 15% contained mutations conferring resistance only to macrolides, and 6% contained mutations conferring resistance to both classes of antibiotics. In C. jejuni, these values were 31% and 0% for quinolone and macrolide resistance, respectively. The rpoB sequence allowed phylogenetic differentiation between C. coli and C. jejuni, which was not possible by 16S rRNA gene analysis. An online Integrated Database Network System (SmartGene, Zug, Switzerland)-based platform for MLST data analysis specific to Campylobacter was implemented. This Web-based platform allowed automated allele and ST designation, as well as epidemiological analysis of data, thus streamlining and facilitating the analysis workflow. Data networking facilitates the exchange of information between collaborating centers. The described approach simplifies and improves the genotyping of Campylobacter, allowing cost-and time-efficient routine monitoring.

Genotypes and Antibiotic Resistances of Campylobacter jejuni and Campylobacter coli Isolates from Domestic and Travel-Associated Human Cases

“…As with previous investigations, flaB typing was poorly congruent with MLST (17,28). Thus, a combination of MLST and flaB typing increased discrimination, as reflected in a higher Simpson's index of diversity: 0.970 compared to 0.928 for MLST and 0.921 for flaB typing alone.…”

“…The C. jejuni isolates showed much higher diversity than the C. coli isolates, as indicated by the distribution of the strains within different clonal complexes (CCs). Of the investigated C. coli strains, 95.0% could be assigned to CC828, confirming a higher clonality within this species (17,24,26).…”

“…In contrast to the previously described protocol (17), 5ϫ FIREPol master mix ready to load (Solis BioDyne, Tartu, Estonia) was used. This led to the following composition for each 30.0-l multiplex PCR: 0.4 M appropriate primer, 1ϫ FIREPol master mix ready to load, 1.0 l lysate (approximately 30 ng DNA), and the addition of MgCl 2 up to a final concentration of 5 mM.…”

“…Multilocus sequence typing (MLST), flaB typing, and genetic determination of resistance to quinolones and macrolides, the two main groups of antibiotics used for treatment of severe cases, based on the sequencing of gyrA and 23S rRNA gene fragments, respectively, were performed as described previously (2,5,6,17,19). In contrast to the previously described protocol (17), 5ϫ FIREPol master mix ready to load (Solis BioDyne, Tartu, Estonia) was used.…”

Broilerlerden İzole Edilen Termofilik Campylabacter’lerin Genotipik, Antimikrobiyal Direnç ve Virulens Profilleri

“…PCR amplification of fragments of flaA and flaB genes comprising the SVRs were performed following the procedures described by Lévesque et al [24] and Korczak et al [25] , respectively. Primers described by those authors were used, amplifying regions of 641 base pairs (bp) and 602 bp of the flaA and flaB genes, respectively.…”