Investigating Major Recurring Campylobacter jejuni Lineages in Luxembourg Using Four Core or Whole Genome Sequencing Typing Schemes

Abstract

Campylobacter jejuni is the leading cause of bacterial gastroenteritis, which has motivated the monitoring of genetic profiles circulating in Luxembourg since 13 years. From our integrated surveillance using a genotyping strategy based on an extended MLST scheme including gyrA and porA markers, an unexpected endemic pattern was discovered in the temporal distribution of genotypes. We aimed to test the hypothesis of stable lineages occurrence by implementing whole genome sequencing (WGS) associated with comprehensive and internationally validated schemes. This pilot study assessed four WGS-based typing schemes to classify a panel of 108 strains previously identified as recurrent or sporadic profiles using this in-house typing system. The strain collection included four common lineages in human infection (N = 67) initially identified from recurrent combination of ST-gyrA-porA alleles also detected in non-human samples: veterinary (N = 19), food (N = 20), and environmental (N = 2) sources. An additional set of 19 strains belonging to sporadic profiles completed the tested panel. All the strains were processed by WGS by using Illumina technologies and by applying stringent criteria for filtering sequencing data; we ensure robustness in our genomic comparison. Four typing schemes were applied to classify the strains: (i) the cgMLST SeqSphere+ scheme of 637 loci, (ii) the cgMLST Oxford scheme of 1,343 loci, (iii) the cgMLST INNUENDO scheme of 678 loci, and (iv) the wgMLST INNUENDO scheme of 2,795 loci. A high concordance between the typing schemes was determined by comparing the calculated adjusted Wallace coefficients. After quality control and analyses with these four typing schemes, 60 strains were confirmed as members of the four recurrent lineages regardless of the method used (N = 32, 12, 7, and 9, respectively). Our results indicate that, regardless of the typing scheme used, epidemic or endemic signals were detected as reflected by lineage B (ST2254-gyrA9-porA1) in 2014 or lineage A (ST19-gyrA8-porA7), respectively. These findings support the clonal expansion of stable genomes in Campylobacter population exhibiting a multi-host profile and accounting for the majority of clinical strains isolated over a decade. Such recurring genotypes suggest persistence in reservoirs, sources or environment, emphasizing the need to investigate their survival strategy in greater depth.

Keywords: Campylobacter jejuni; WGS typing scheme comparison; clones; core genome MLST; recurring genotypes; typing schemes; whole genome MLST; whole genome sequencing.

Figure 1

Distribution of strains occurrence for lineages A to D over time. Clinical strains of the laboratory collection are displayed in gray (extended MLST typing). Colors represent to source of selected isolates that were analyzed by WGS: human (red), cattle and sheep (green), poultry (yellow), and surface water (blue) samples.

Figure 2

Shared targets between the three compared schemes: cgMLST SeqSphere+ (637 targets), cgMLST INNUENDO (678 targets) and cgMLST Oxford (1,343 targets) highlighted as set sizes. The central bars represent the number of shared or unique targets in or between the different schemes. The points below define the members of the respective sets. For example, 432 targets are present in all three cgMLSTs (SeqSphere+, Oxford, and INNUENDO) and 243 targets are present in both the cgMLSTs Oxford and INNUENDO but not in the cgMLST SeqSphere+. For an overview of shared targets, also refer to Supplementary Data S4 .

Figure 3

Venn diagram showing the relationship between the loci identified in the four lineages by wgMLST INNUENDO analysis (2,795 targets). A total of 995, 975, 1,000, and 925 targets were detected in lineages A, B, C, and D, respectively.

Figure 4

Minimum Spanning Trees generated using PHYLOViZ for (A) cgMLST SeqSphere+ (cut-off: 13), (B) cgMLST and accessory targets SeqSphere+ (cut-off: 13), (C) cgMLST INNUENDO (cut-off: 4), (D) cgMLST Oxford (cut-off defined by dynamic core analysis: 11) and (E) wgMLST INNUENDO (cut-off defined by dynamic core analysis: 9) analyses on tool. Lineage A is displayed in blue, lineage B in red, lineage C in orange, and lineage D in green and unique combinations in gray.