Hyperglycemia Increases Severity of Staphylococcus aureus Osteomyelitis and Influences Bacterial Genes Required for Survival in Bone

Abstract

Hyperglycemia, or elevated blood glucose, renders individuals more prone to developing severe Staphylococcus aureus infections. S. aureus is the most common etiological agent of musculoskeletal infection, which is a common manifestation of disease in hyperglycemic patients. However, the mechanisms by which S. aureus causes severe musculoskeletal infection during hyperglycemia are incompletely characterized. To examine the influence of hyperglycemia on S. aureus virulence during invasive infection, we used a murine model of osteomyelitis and induced hyperglycemia with streptozotocin. We discovered that hyperglycemic mice exhibited increased bacterial burdens in bone and enhanced dissemination compared to control mice. Furthermore, infected hyperglycemic mice sustained increased bone destruction relative to euglycemic controls, suggesting that hyperglycemia exacerbates infection-associated bone loss. To identify genes contributing to S. aureus pathogenesis during osteomyelitis in hyperglycemic animals relative to euglycemic controls, we used transposon sequencing (TnSeq). We identified 71 genes uniquely essential for S. aureus survival in osteomyelitis in hyperglycemic mice and another 61 mutants with compromised fitness. Among the genes essential for S. aureus survival in hyperglycemic mice was the gene encoding superoxide dismutase A (sodA), one of two S. aureus superoxide dismutases involved in detoxifying reactive oxygen species (ROS). We determined that a sodA mutant exhibits attenuated survival in vitro in high glucose and in vivo during osteomyelitis in hyperglycemic mice. SodA therefore plays an important role during growth in high glucose and promotes S. aureus survival in bone. Collectively, these studies demonstrate that hyperglycemia increases the severity of osteomyelitis and identify genes contributing to S. aureus survival during hyperglycemic infection.

Keywords: Staphylococcus aureus; hyperglycemia; osteomyelitis; superoxide dismutase; transposon sequencing.

FIG 1

Acute hyperglycemia increases S. aureus burdens and dissemination during osteomyelitis. Eight-week-old male mice were treated with sodium citrate (vehicle) or streptozotocin (STZ) intraperitoneally for 5 days. Ten days after the final injection, mice were infected with 1 × 10⁶ CFU of WT S. aureus via intraosseous injection. (A) Blood glucose concentration was quantified from a tail vein bleed immediately prior to inoculation (day 0) and on the day of sacrifice (day 14). The dotted line indicates the hyperglycemia threshold of 250 mg/dL. (B to F) Mice were sacrificed at day 14 postinfection, and the bacterial burdens (CFU) were enumerated in infected femur (B), contralateral femur (C), kidneys (D), liver (n = 4 mice per group) (E), and heart (n = 4 mice in STZ-treated group) (F). One experiment was conducted with n = 5 mice per group unless otherwise noted. Dotted lines indicate limit of detection. Horizontal lines indicate means, and error bars represent SD. Significance was determined with the Mann-Whitney test (B to F). *, P < 0.05; **, P < 0.01.

FIG 2

Chronic hyperglycemia increases S. aureus burdens and dissemination during osteomyelitis. Eight-week-old male mice were treated with sodium citrate (vehicle) or STZ intraperitoneally for 5 days. Thirty days after the final injection, mice were infected with 1 × 10⁶ CFU of WT S. aureus via intraosseous injection. (A) Blood glucose concentration was quantified from a tail vein bleed immediately prior to inoculation (day 0) and on the day of sacrifice (day 14). The dotted line indicates the hyperglycemia threshold of 250 mg/dL. (B to F) Mice were sacrificed at day 14 postinfection, and the bacterial burdens (CFU) were enumerated in infected femur (B), contralateral femur (C), kidneys (D), liver (E), and heart (F). One experiment was conducted with n = 5 mice per group. Dotted lines indicate limit of detection. Horizontal lines indicate means, and error bars represent SD. Significance was determined with the Mann-Whitney test (B to F). *, P < 0.05; **, P < 0.01.

FIG 3

S. aureus incites greater bone destruction in acute hyperglycemic animals. Eight-week-old male mice were treated with sodium citrate (vehicle) or STZ intraperitoneally for 5 days. Ten days after the final injection, mice were infected with 1 × 10⁵ CFU of WT S. aureus via intraosseous injection. At 14 days postinfection, the infected femur and contralateral femur were isolated for microcomputed tomography. Cortical bone loss in infected femurs (A), trabecular bone volume divided by total volume (BV/TV) of infected femurs (B), and BV/TV of infected femurs relative to contralateral femurs (C) were quantified. Trabecular number (Tb.N) (D), trabecular thickness (Tb.Th) (E), and trabecular spacing (Tb.Sp) (F) were quantified in infected femurs. One experiment was conducted with n = 5 mice per group. Horizontal lines indicate means, and error bars represent SD. Significance was determined with the Mann-Whitney test. *, P < 0.05; **, P < 0.01.

FIG 4

Transposon sequencing reveals genes essential for S. aureus survival during osteomyelitis in mice with hyperglycemia. Eight-week-old male mice were treated with sodium citrate (vehicle) or STZ intraperitoneally for 5 days. Ten days after the final injection, mice were infected with 5 × 10⁶ CFU of the S. aureus TnSeq library via intraosseous injection. At day 4 postinfection, bacteria were recovered and Illumina sequencing was used to identify the abundance of S. aureus mutants under each condition. (A) Based on Dval calculations, the number of essential genes (Dval < 0.01) in vitro in BHI, in vivo in STZ-treated hyperglycemic mice, and in vivo in euglycemic vehicle-treated mice were enumerated. (B) The annotated genes essential for S. aureus survival (Dval < 0.01) and the mutants with compromised fitness (Dval > 0.01 and Dval < 0.1) only under the condition of osteomyelitis during hyperglycemia were organized into categories of metabolism, gene regulation, virulence, DNA repair, cell signaling, and peptidoglycan (PG) synthesis. Two experiments were conducted with 6 mice per in vivo group per experiment, pooled in samples of 2 for n = 6 in sequencing analysis.

FIG 5

S. aureus sodA is required for survival during culture in high glucose. (A and B) WT, sodA::tet, and sodM::erm S. aureus strains were grown in 10 mL of TSB with and without 500 mg/dL of added glucose in flasks covered with foil (aerobic) (A) or capped (microaerobic) (B) with shaking at 37°C. CFU were quantified every 24 h over the course of 5 days and normalized to time zero. (C) pH was measured every 24 h over the course of 5 days in TSB with 500 mg/dL of glucose under aerobic and microaerobic conditions. (D) WT, sodA::tet, and sodA::tet attC::sodA S. aureus strains were grown in 10 mL of TSB and TSB with 500 mg/dL of glucose in flasks covered with foil (aerobic) (n = 2 technical replicates and n = 3 biological replicates). The line represents the mean, and error bars represent SD (A, B, and D). Significance was determined with two-way ANOVA and Dunnett’s multiple-comparison test. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (sodA::tet TSB plus glucose). &, P < 0.05; &&, P < 0.01 (WT TSB plus glucose). ^, P < 0.05; ^^, P < 0.01 (sodM::erm TSB plus glucose). #, P < 0.05 (sodA::tet attC::sodA TSB plus glucose). $, P < 0.01 (sodA::tet TSB). All comparisons were made to the WT in TSB.

FIG 6

SodA is important for S. aureus survival during osteomyelitis in hyperglycemic mice. Eight-week-old male mice were treated with STZ intraperitoneally for 5 days. Ten days after the final injection, mice were infected with 1 × 10⁵ CFU of WT, sodM::erm, sodA::tet, and sodA::tet sodM::erm S. aureus strains via intraosseous injection. (A) Weights were recorded every 24 h and normalized to the starting weight of each animal on the day of infection (percent weight). (B to E) Mice were sacrificed at day 14 postinfection, and the bacterial burdens (CFU) were enumerated in infected femur (B), kidneys (C), liver (D), and heart (E). Different shapes indicate data collected from 3 distinct experiments (n = 10 mice per group). Dotted lines indicate limit of detection. Horizontal lines indicate means, and error bars represent SD. Significance was determined with two-way ANOVA and Dunnett’s multiple-comparison test (A) and Kruskal-Wallis with Dunn’s multiple-comparison test (B to E). *, P < 0.05; **, P < 0.01; ***, P < 0.001. All comparisons were made to the WT.

FIG 7

SodA is not necessary for S. aureus survival during osteomyelitis in euglycemic mice. Eight-week-old male mice were treated with sodium citrate (vehicle) intraperitoneally for 5 days. Ten days after the final injection, mice were infected with 1 × 10⁵ CFU of WT, sodA::tet, and sodM::erm S. aureus strains via intraosseous injection. (A) Weights were recorded every 24 h and normalized to the starting weight of each animal on the day of infection (percent weight). (B) Mice were sacrificed at day 14 postinfection, and the bacterial burdens (CFU) were enumerated in infected femurs. Different shapes indicate data collected from 3 distinct experiments (n = 14 mice per group). The dotted line indicates limit of detection. Horizontal lines indicate means, and error bars represent SD. Significance was determined with two-way ANOVA and Dunnett’s multiple-comparison test (A) and Kruskal-Wallis with Dunn’s multiple-comparison test (B). *, P < 0.05. All comparisons were made relative to WT.