Myo-inositol utilization by Citrobacter koseri promotes brain infection.

Citrobacter species are opportunistic bacterial pathogens that are implicated in both nosocomial and community-acquired infections. Among the Citrobacter species, Citrobacter koseri is often isolated from clinical material, and it can cause meningitis and brain abscesses in neonates and immunocompromised individuals, thus posing a great threat to human health. However, the virulence determinants of C. koseri remain largely unknown. Myo-inositol is an abundant carbohydrate in the environment and in certain organs of the human body, especially the brain. The C. koseri genome harbors a cluster of genes, QCQ70420.1 to QCQ70429.1 (named the Ino-cluster in this study), which encode IolBCDE, MmsA, and an ATP-binding cassette transporter. The gene cluster may be involved in the utilization of myo-inositol. To investigate the functions of the Ino-cluster in C. koseri, we constructed a mutant strain by deleting the Ino-cluster and found that the mutant could not use myo-inositol as the sole carbon source, confirming that this cluster is responsible for myo-inositol utilization. Moreover, we investigated the function of the Ino-cluster and myo-inositol utilization in C. koseri pathogenicity. Deletion of the Ino-cluster significantly impaired C. koseri colonization of the brain of infected Sprague-Dawley (SD) rats and BALB/c mice, and this increased the survival rate of the infected animals, indicating that the Ino-cluster and the ability to use myo-inositol are essential for C. koseri pathogenicity. Taken together, our findings suggest that using the Ino-cluster products, C. koseri can exploit the abundant myo-inositol in the brain as a carbon source for growth, thus promoting colonization and virulence.

Identical bacterial populations colonize premature infant gut, skin, and oral microbiomes and exhibit different in situ growth rates.

The initial microbiome impacts the health and future development of premature infants. Methodological limitations have led to gaps in our understanding of the habitat range and subpopulation complexity of founding strains, as well as how different body sites support microbial growth. Here, we used metagenomics to reconstruct genomes of strains that colonized the skin, mouth, and gut of two hospitalized premature infants during the first month of life. Seven bacterial populations, considered to be identical given whole-genome average nucleotide identity of >99.9%, colonized multiple body sites, yet none were shared between infants. Gut-associated Citrobacter koseri genomes harbored 47 polymorphic sites that we used to define 10 subpopulations, one of which appeared in the gut after 1 wk but did not spread to other body sites. Differential genome coverage was used to measure bacterial population replication rates in situ. In all cases where the same bacterial population was detected in multiple body sites, replication rates were faster in mouth and skin compared to the gut. The ability of identical strains to colonize multiple body sites underscores the habit flexibility of initial colonists, whereas differences in microbial replication rates between body sites suggest differences in host control and/or resource availability. Population genomic analyses revealed microdiversity within bacterial populations, implying initial inoculation by multiple individual cells with distinct genotypes. Overall, however, the overlap of strains across body sites implies that the premature infant microbiome can exhibit very low microbial diversity.

Iron induces bimodal population development by Escherichia coli.

Bacterial biofilm formation is a complex developmental process involving cellular differentiation and the formation of intricate 3D structures. Here we demonstrate that exposure to ferric chloride triggers rugose biofilm formation by the uropathogenic Escherichia coli strain UTI89 and by enteric bacteria Citrobacter koseri and Salmonella enterica serovar typhimurium. Two unique and separable cellular populations emerge in iron-triggered, rugose biofilms. Bacteria at the air-biofilm interface express high levels of the biofilm regulator csgD, the cellulose activator adrA, and the curli subunit operon csgBAC. Bacteria in the interior of rugose biofilms express low levels of csgD and undetectable levels of matrix components curli and cellulose. Iron activation of rugose biofilms is linked to oxidative stress. Superoxide generation, either through addition of phenazine methosulfate or by deletion of sodA and sodB, stimulates rugose biofilm formation in the absence of high iron. Additionally, overexpression of Mn-superoxide dismutase, which can mitigate iron-derived reactive oxygen stress, decreases biofilm formation in a WT strain upon iron exposure. Not only does reactive oxygen stress promote rugose biofilm formation, but bacteria in the rugose biofilms display increased resistance to H(2)O(2) toxicity. Altogether, we demonstrate that iron and superoxide stress trigger rugose biofilm formation in UTI89. Rugose biofilm development involves the elaboration of two distinct bacterial populations and increased resistance to oxidative stress.

Microglial activation by Citrobacter koseri is mediated by TLR4- and MyD88-dependent pathways.

Citrobacter koseri is a Gram-negative bacterium that can cause a highly aggressive form of neonatal meningitis, which often progresses to establish multifocal brain abscesses. Despite its tropism for the brain parenchyma, microglial responses to C. koseri have not yet been examined. Microglia use TLRs to recognize invading pathogens and elicit proinflammatory mediator expression important for infection containment. In this study, we investigated the importance of the LPS receptor TLR4 and MyD88, an adaptor molecule involved in the activation of the majority of TLRs in addition to the IL-1 and IL-18 receptors, for their roles in regulating microglial activation in response to C. koseri. Proinflammatory mediator release was significantly reduced in TLR4 mutant and MyD88 knockout microglia compared with wild-type cells following exposure to either live or heat-killed C. koseri, indicating a critical role for both TLR4- and MyD88-dependent pathways in microglial responses to this pathogen. However, residual proinflammatory mediator expression was still observed in TLR4 mutant and MyD88 KO microglia following C. koseri exposure, indicating a contribution of TLR4- and MyD88-independent pathway(s) for maximal pathogen recognition. Interestingly, C. koseri was capable of surviving intracellularly in both primary microglia and macrophages, suggesting that these cells may serve as a reservoir for the pathogen during CNS infections. These results demonstrate that microglia respond to C. koseri with the robust expression of proinflammatory molecules, which is dictated, in part, by TLR4- and MyD88-dependent signals.

Citrobacter koseri brain abscess in the neonatal rat: survival and replication within human and rat macrophages.

A unique feature of Citrobacter koseri is the extremely high propensity to initiate brain abscesses during neonatal meningitis. Previous clinical reports and studies on infant rats have documented many Citrobacter-filled macrophages within the ventricles and brain abscesses. It has been hypothesized that intracellular survival and replication within macrophages may be a mechanism by which C. koseri subverts the host response and elicits chronic infection, resulting in brain abscess formation. In this study, we showed that C. koseri causes meningitis and brain abscesses in the neonatal rat model, and we utilized histology and magnetic resonance imaging technology to visualize brain abscess formation. Histology and electron microscopy (EM) revealed that macrophages (and not fibroblasts, astrocytes, oligodendrocytes, or neurons) were the primary target for long-term C. koseri infection. To better understand C. koseri pathogenesis, we have characterized the interactions of C. koseri with human macrophages. We found that C. koseri survives and replicates within macrophages in vitro and that uptake of C. koseri increases in the presence of human pooled serum in a dose-dependent manner. EM studies lend support to the hypothesis that C. koseri uses morphologically different methods of uptake to enter macrophages. FcgammaRI blocking experiments show that this receptor primarily facilitates the entry of opsonized C. koseri into macrophages. Further, confocal fluorescence microscopy demonstrates that C. koseri survives phagolysosomal fusion and that more than 90% of intracellular C. koseri organisms are colocalized within phagolysosomes. The ability of C. koseri to survive phagolysosome fusion and replicate within macrophages may contribute to the establishment of chronic central nervous system infection including brain abscesses.

Myocarditis in sibling boxer puppies associated with Citrobacter koseri infection.

Two sibling Boxer puppies presented with severe suppurative myocarditis in the absence of additional disseminated suppurative foci. The identification of gram-negative bacteria within areas of myocarditis in both puppies and the pure growth of large numbers of Citrobacter koseri from the myocardial lesions in one of the dogs were consistent with a bacterial etiology. The fact that C. koseri is an opportunist pathogen suggested intercurrent immunosuppression. The finding of a concomitant bacterial myocarditis in two canine siblings is novel. The case is also unusual in that syncope could be related to the myocardial injury.