Unraveling Citrobacter koseri Infection Outbreaks Using Next-generation Sequencing: A Clinical Case Study

Citrobacter koseri (C.koseri) is a normal gut microbe but can cause severe opportunistic infections in immunocompromised individuals and neonates.Due to sudden hospital outbreaks, it has recently gained attention.The lack of adequate environmental surveillance and poor understanding of the epidemiology of its spread warrant the use of more sensitive and superior analysis methods.Next-generation sequencing is a powerful and rapidly emerging tool yet to be validated for routine molecular diagnosis and epidemiology of C. koseri infections.In this study, the next generation sequencing (NGS) was used to investigate the outbreak of 4 cases of C. koseri bloodstream infection in a Neurology setting within a hospital over three days.The blood samples were tested for bacterial culture among the patients with fever (body temperature ≥38°C) and chills.Additionally, the environmental samples were also cultured.A total of 4 patients with chills and fever within three days of admission and cases of sepsis due to C. koseri were identified through routine diagnosis.C. koseri was also found in the sealing fluid of environmental samples.NGS analysis was conducted on four bacterial samples from the patient's blood.The NGS data showed that entire paired-end reads were assembled into a 4.5 Mb genome with an average GC content 54.66%.The phylogram is based on the global pan-genome, suggesting a distinct clade in the identified samples.Phylogenetic analysis of the 16S RNA showed two distinct clusters.Cluster 1 originated from CKB211, while Cluster 2 was isolated from patients in the same ward (CKB212, CKB213, and CKB214).The core-pan evolutionary analysis indicated that CKB211 had a distant evolutionary relationship with other strains and more subtle evolutionary relationships were also analyzed.This analysis is consistent with the physical distance of these patients.It strongly indicates a likely route of infection via shared saline, which was the common operational approach.The study provides unique insights into the rare infection caused by C. koseri, utilizing NGS and phylogenetic analysis.In the present study, although both 16S RNA and the core-pan phylogenetic tree can be used for the evolutionary analysis of C. koseri, the core-pan analysis involves a greater sequence and provides a more nuanced understanding of divergence.The study suggests using core-pan for the evolutionary analysis of C. koseri.However, considering the limited sample size in this study, the applicability of this method remains to be explored.