Abstract
Citrobacter species are opportunistic bacterial pathogens that have been implicated in both nosocomial and community-acquired infections. Among the genus Citrobacter, Citrobacter koseri is often isolated from clinical material, and has been known to cause meningitis and brain abscess in neonates and immunocompromised individuals. The virulence determinants of Citrobacter, however, remain largely unknown. Based on traditional methods, the genus Citrobacter has been divided into 11 species, but this has been problematic. Here, we determined an improved, detailed, and more accurate phylogeny of the genus Citrobacter based on whole genome sequence (WGS) data from 129 Citrobacter genomes, 31 of which were sequenced in this study. A maximum likelihood (ML) phylogeny constructed with core genome single-nucleotide polymorphisms (SNPs) classified all Citrobacter isolates into 11 distinct groups, with all C. koseri strains clustering into a single group. For comprehensive and systematic comparative genomic analyses, we investigated the distribution of virulence factors, resistance genes, and macromolecular secretion systems among the Citrobacter genus. Moreover, combined with group-specific genes analysis, we identified a key gene cluster for iron transport, which is present in the C. koseri group, but absent in other the groups, suggesting that the high-pathogenicity island (HPI) cluster may be important for the pathogenicity of C. koseri. Animal experiments showed that loss of the HPI cluster significantly decreased C. koseri virulence in mice and rat. Further, we provide evidence to explain why Citrobacter freundii is less susceptible than C. koseri to several antibiotics in silico. Overall, our data reveal novel virulence clusters specific to the predominantly pathogenic C. koseri strains, which form the basis for elucidating the virulence mechanisms underlying these important pathogens.
Highlights
MATERIALS AND METHODSThe Citrobacter genus belongs to the Enterobacteriaceae family, which is a distinct group of aerobic, Gram-negative, nonspore-forming, rod-shaped bacteria that typically utilize citrate as their primary carbon source (Janda et al, 1994; Sakazaki, 1997)
We found that all species had genes which encoded different types of antibiotic efflux pumps, including resistance-nodulation-cell division (RND) types, majorfacilitator superfamily (MFS) types, ATP-binding cassette (ABC) types, which confer resistant to aminoglycoside antibiotics, aminocoumarin antibiotics, fluoroquinolone, lincosamide antibiotics, cephalosporin, cephamycin, and penam (Supplementary Table S4)
We found that the well characterized high-pathogenicity island (HPI) in Yersinia pestis CO92 was closely related to the HPI cluster in C. koseri TBCP-5362, and that these gene clusters are present in similar genomic locations and encode proteins with more than 95% sequence identity
Summary
MATERIALS AND METHODSThe Citrobacter genus belongs to the Enterobacteriaceae family, which is a distinct group of aerobic, Gram-negative, nonspore-forming, rod-shaped bacteria that typically utilize citrate as their primary carbon source (Janda et al, 1994; Sakazaki, 1997). Citrobacter infections usually supervene upon debilitated, hospitalized patients, with multiple comorbidities (Gross et al, 1973; Williams et al, 1984; Kline and Kaplan, 1987; Mohanty et al, 2007; Samonis et al, 2009). Urinary tract infections caused by Citrobacter account for approximately half of all infections, there are no reports of statistically significant associations between infection sites and Citrobacter species, except for C. koseri, which exhibits a remarkable degree of tropism for the brain (Gross et al, 1973; Ribeiro et al, 1976; Curless, 1980; Williams et al, 1984; Kline and Kaplan, 1987; Doran, 1999)
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