Essentiality of the virulence plasmid-encoded factors in disease pathogenesis of the major lineage of hypervirulent Klebsiella pneumoniae varies in different infection niches

Abstract

Background: Hypervirulent Klebsiella pneumoniae (HvKp) can metastasise to extra-intestinal sites to cause disseminated disease such as pyogenic liver abscesses. HvKp harbours a large virulence plasmid (KpVP) that contributes to pathogenicity. We previously identified a crucial gene region that confers virulence in SGH10 (ST23, K1 capsule), spanning genes encoding the siderophores aerobactin and salmochelin, as well as the regulator of mucoidy phenotype A (iuc-rmp-iro).

Methods: SGH10 isogenic mutants of aerobactin, rmpA, and salmochelin were generated and tested in vitro for their siderophore production, hypermucoviscosity and growth. We investigated the essentiality of these factors in different murine infection or colonisation models.

Findings: In a lung pneumonia model, capsule modulation by rmpA was the primary driver of high bacterial burden in the lung. In a systemic infection setting, rmpA was still the primary driver, followed by a significant contribution by salmochelin, that conferred virulence. However, the role of aerobactin was more significant in hvKp persistence in the gut. We further examined a large collection of Kp genomes and observed that the iro loci is often co-inherited with iuc in KpVP-1, suggesting the evolutionary importance of expressing both siderophores in these lineages.

Interpretation: HvKp typically colonises the intestinal niche, however, the acquisition of the KpVP plasmid has enabled it to thrive outside the gut and cause metastatic infections. While the iuc-rmp-iro region is pivotal in bestowing virulence, the encoded factors contribute differently to the success of the pathogen in various infection sites, where the microenvironment, nutrient availability and immune response can vary. Thus, our study demonstrates that possessing the iuc-rmp-iro gene region can be an evolutionary advantage by allowing for flexibility in modulating siderophore and capsule expression in order for K. pneumoniae to thrive in distinct host niches.

Funding: This work is funded by the National Research FoundationMOH-000925-00 to YH Gan and OFYIRG22jul-0042 by the National Medical Research Council (NMRC) to THT.

Keywords: Hypervirulent; Klebsiella pneumoniae; Liver abscess; Plasmid; Siderophore.

Fig. 1

SGH10 predominantly produces aerobactin, enterobactin, and enterobactin-derived siderophores. Siderophore quantification of SGH10 and its mutants cultured in a) DMEM + 10% FBS, b) MES-buffered DMEM (maintained at pH 7), c) DMEM supplemented with 200 uM 2-2′Bipyridyl (BIP) and d) 25% mouse serum. Hypermucoviscosity quantification for SGH10 and mutants cultured overnight in e) LB media or f) DMEM + 10% FBS with capsule-null ΔwcaJ as a negative control. Data was analysed by one-way ANOVA. Bar graphs represent Mean ± SD, each dot represents one biological replicate.

Fig. 2

rmpA is responsible for bacterial growth in the pneumonia model. a) CFU burden in the lungs were enumerated at 48 hpi following intranasal administration with 10³ of SGH10 and its mutants. b) Kaplan–Meier survival curve of mice infected via intranasal administration with 10³ CFU of SGH10 and its mutants. c) CFU burden in lungs of mice at 48 hpi following intranasal administration with 10² CFU of SGH10 and its mutants. Where no colony was enumerated at the limit of detection, data was plotted as 1 on log axis. Means are represented. Data was analysed by Kruskal–Wallis test. Each dot represents one mouse. Statistical comparison was performed by Gehan-Breslow-Wilcoxon test between wild-type and each mutant. Data comprises n = 4–5 mice per group.

Fig. 3

rmpA and salmochelin contribute to virulence in systemic infection. a–d) Mice were infected via intraperitoneal injection with 10³–10⁶ CFU of SGH10 and its mutants. Kaplan–Meier survival curves were plotted, and statistical comparison was performed by Gehan-Breslow-Wilcoxon test between wild-type and each mutant. Experiments were conducted with n = 5–16 mice per group. e–h) Mice were infected via intraperitoneal injection with 10⁴ CFU of SGH10 and its mutants, and CFU burden in the blood, spleen, lungs and liver were enumerated at 12 hpi. Where no colony was enumerated at the limit of detection, data was plotted as 1 on log axis. Data was analysed by Kruskal–Wallis multiple comparisons test. Each dot represents one mouse, data was pooled from n = 2 experiments. Means are represented. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.01; ∗∗∗∗p < 0.0001.

Fig. 4

Aerobactin is pivotal for long term gut colonisation by SGH10. Mice were pre-treated with ampicillin for three days prior to colonisation with SGH10 and its isogenic mutants by oral gavage. CFU counts were enumerated from shed stool pellets to track colonisation loads of SGH10 against its a)KpVP-cured mutant (n = 5 mice) and b) its KpVP gene mutants (n = 4–5 mice). c) Gut colonisation CFU data from b were Log₁₀ transformed and area under the curve (AUC) calculated for SGH10 and its mutants between 5 and 14 dpi for each mouse. Data from 1 to 3 dpi was excluded due to the high colonisation loads consistent among all strains that would skew the total area calculation and make differences at later timepoints indistinguishable as colonisation loads decrease. d) Gut colonisation loads of mice colonised with SGH10 and its capsule-null ΔwcaJ mutant (n = 4–5 mice). Data in a, b, and d are represented by geometric mean ± SD. Data in b was analysed with 2-way ANOVA and Dunnett's multiple comparison test. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001. Data in c was analysed by Kruskal–Wallis multiple comparisons test, Mean ± SD are represented.

Fig. 5

Iro and iuc loci are commonly co-inherited in KpVP-1 harbouring hypervirulent Kp strains. Presence and absence of iuc1 and iro1 in 3928 Klebsiella pneumoniae genomes with the KpVP-1 virulence plasmid, categorised by a) sublineages and b) sample type or source. Sublineages are defined on the basis of the 629-loci cgMLST scheme and associated cgLIN code nomenclature. The size of the circles correspond to genome counts corresponding to each category. Hypervirulent and MDR sublineages, and samples sourced from patients/humans are indicated.