Assessing mutation accumulation in DNA repair-deficient Listeria monocytogenes: implications for cgMLST cluster thresholds in outbreak analysis

Abstract

Background: Listeria (L.) monocytogenes is primarily transmitted via contaminated food and can cause listeriosis, an infection often associated with sepsis and meningitis in at-risk individuals. Accurate outbreak detection relies on whole genome sequencing (WGS) and core genome multilocus sequence typing (cgMLST), which use allele thresholds to identify related strains.

Methods: This study investigated mutation rates in L. monocytogenes, focusing on isolates with DNA repair deficiencies. Serial subcultivations were performed, comparing a repair-deficient isolate with a wild-type control. Genetic variability was assessed using WGS and cgMLST.

Results: Mutation rates were significantly higher in repair-deficient isolates, exceeding typical cgMLST thresholds currently used in Listeria outbreak investigations, leading to a misclassification of related isolates as unrelated. An additional analysis of the Austrian Listeria database revealed that such deficiencies are rare among isolates.

Conclusions: The standard 7-allele cgMLST threshold effectively identifies related strains in most cases, but may require adjustments for hypermutator strains. Incorporating DNA repair data could improve the accuracy of outbreak investigations, ensuring reliable public health responses.

Keywords: Listeria monocytogenes; allele threshold; core genome multilocus sequence typing; listeriosis; mutation rate; outbreak detection.

Figure 1

Minimum spanning tree based on the cgMLST allelic profiles of subcultures 4, 6, 8, 10, 12 and 14 of the wild-type and mutant isolate. Each circle represents a given allelic profile. The number on the connecting lines illustrates the number of differing alleles; MI, mutant isolate, WT, wild-type isolate, 37°C: aerobic incubation at 37°C for 48 hours, 4-8°C: aerobic incubation at 4-8°C for 48 hours, 37°C + P: aerobic incubation at 37°C for 48 hours with the addition of penicillin discs.