Comparative genomic analyses of virulence and antimicrobial resistance in Citrobacter werkmanii, an emerging opportunistic pathogen

Authors: AGUIRRE-SANCHEZ, JOSE - Center For Research In Food And Development (CIAD); Quinones, Beatriz; ORTIZ-MUÑOZ, JOSE - Autonomous University Of Sinaloa; PRIETO-ALVARADO, ROGELIO - Autonomous University Of Sinaloa; VEGA-LOPEZ, INES - Autonomous University Of Sinaloa; MARTINEZ-URTAZA, JAIME - Autonomous University Of Barcelona; Lee, Bertram; CHAIDEZ, CRISTOBAL - Center For Research In Food And Development (CIAD)

Submitted to: Microorganisms
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/13/2023
Publication Date: 8/19/2023
Citation: Aguirre-Sanchez, J.R., Quinones, B., Ortiz-Muñoz, J.A., Prieto-Alvarado, R., Vega-Lopez, I.F., Martinez-Urtaza, J., Lee, B.G., Chaidez, C. 2023. Comparative genomic analyses of virulence and antimicrobial resistance in Citrobacter werkmanii, an emerging opportunistic pathogen. Microorganisms. 11(8). Article 2114. https://doi.org/10.3390/microorganisms11082114.
DOI: https://doi.org/10.3390/microorganisms11082114

Interpretive Summary: Citrobacter species are gram-negative bacteria and are found in many reservoirs, including the human and animal gastrointestinal tracts as well as in water, soil, and food. Citrobacter is considered an opportunistic pathogen and has been implicated as a causative agent of hospital settings and community-acquired infections among immunocompromised patients and neonates. Several transmission routes have been proposed for this bacterial pathogen such as fecal-oral, contaminated food, hospital equipment, and person to person transmission. Among the Citrobacter species, Citrobacter freundii and Citrobacter koseri have been implicated in causing most of the opportunistic human infections. Recent evidence has demonstrated that Citrobacter werkmanii is an emerging opportunistic pathogen in developing countries since this species has been linked as a causative agent of wound infection, urinary tract infections and bacteremia in humans. In the present study, comparative genomic analyses were conducted on C. werkmanii strains recovered from a major agricultural region for horticultural products in Mexico as well as from various geographical locations and sources to identify relevant virulence and antimicrobial resistance genes. This research has identified key determinants implicated in highly pathogenic and multidrug resistance profiles and has provided fundamental information to enable the characterization of C. werkmanii as an emerging opportunistic pathogen.

Technical Abstract: Citrobacter werkmanii is an emerging opportunistic pathogen in developing countries and is a causative agent of wound infection, urinary tract infections and bacteremia in humans. To further characterize C. werkmanii as an opportunistic pathogen, the present study conducted comparative genomic analyses of C. werkmanii strains recovered from a major agricultural region in Northwestern Mexico as well as from various geographical locations and sources to identify relevant virulence and antimicrobial resistance genes. For the recovery of Citrobacter isolates from agricultural river water, a use of a size-exclusion ultrafiltration method with selective media enrichment was employed. Next-generation sequencing was performed on the recovered Citrobacter isolates for genome characterization. Phylogenetic analyses, based on concatenation of the MLST genes fusA, leuS, pyrG and rpoB and also of the housekeeping recN gene, revealed that the river water isolates CW031 and CW032 belong to the species C. werkmanii and were found to cluster together in a separate branch with the reference strain C. werkmanii NBRC105721 as the most closely-related species. Comparative analyses demonstrated that strains CW31 and CW32 harbor genomic islands conferring genetic traits implicated in lipopolysaccharide synthesis, transcriptional/translational regulation, iron transport, temperature stress resistance, arsenic resistance, RND-type multidrug efflux pumps. To further expand on the genetic diversity analysis of the C. werkmanii isolates, a pangenome of 30 publicly available C. werkmanii strains was subsequently constructed. Among the examined C. werkmanii isolates, the core genome was the most abundant with a total of 126,303 genes with functional roles in carbohydrate and general metabolism and transport, cell wall structure, and signal transduction. Moreover, the shell and accessory pangenomes were found to contain 6,832 and 7,442 genes, respectively. The shell genome was found to be mainly composed of genes with a role in secretion, pilus assembly protein, and phage structure genes. By contrast, over 10% of the genes in the accessory genome had unspecified functions. The findings from these comparative genomic analyses have demonstrated C. werkmanii strains harbor highly pathogenic and multidrug resistant genetic profiles, indicating a virulence potential of this commensal as an opportunistic human pathogen.