Table_2_Investigating Major Recurring Campylobacter jejuni Lineages in Luxembourg Using Four Core or Whole Genome Sequencing Typing Schemes.xlsx

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.

空肠弯曲杆菌（Campylobacter jejuni）是细菌性胃肠炎的首要致病菌，这促使卢森堡自13年前起启动针对其流行遗传谱的监测工作。本研究采用包含gyrA与porA标记的扩展多位点序列分型（MLST）方案构建基因分型策略，开展综合监测，期间意外发现基因型的时间分布呈现出地方流行模式。本研究旨在结合全基因组测序（WGS）与经国际验证的综合分型方案，验证"稳定谱系持续流行"这一假说。这项预实验研究评估了4种基于WGS的分型方案，用于对此前通过本实验室自建分型系统鉴定为复发或散发型的108株菌株进行分类。

本次纳入的菌株集合包含67株人类感染病例中常见的4种谱系，这些谱系最初通过ST-gyrA-porA等位基因的复发组合模式鉴定得到，且同时在非人类样本中检出：其中动物源19株、食品源20株、环境源2株。另有19株属于散发型的菌株补充了本次测试的菌株组。

所有菌株均采用Illumina测序技术完成WGS，并应用严格标准对测序数据进行过滤，以确保基因组比较分析的稳健性。本次研究共采用4种分型方案对菌株进行分类：（i）包含637个基因座的核心基因组MLST（cgMLST）SeqSphere+分型方案；（ii）包含1343个基因座的核心基因组MLST（cgMLST）牛津分型方案；（iii）包含678个基因座的核心基因组MLST（cgMLST）INNUENDO分型方案；（iv）包含2795个基因座的全基因组MLST（wgMLST）INNUENDO分型方案。通过对比计算得到的调整华莱士系数，证实各分型方案间具有较高的一致性。

经4种分型方案的质量控制与分析后，无论采用哪种方法，均确认有60株菌株属于4种复发谱系（分别为32株、12株、7株和9株）。研究结果显示，无论使用哪种分型方案，均能检测到流行或地方流行信号：例如2014年检出的谱系B（ST2254-gyrA9-porA1）对应流行信号，而谱系A（ST19-gyrA8-porA7）则对应地方流行信号。上述发现支持空肠弯曲杆菌种群中存在稳定基因组的克隆扩增，这类种群具有多宿主特性，且占十余年间分离到的临床菌株的绝大多数。这类反复出现的基因型提示其在储存宿主、传染源或环境中持续存在，这凸显了深入研究其生存策略的必要性。