Staphylococcus aureus isolates from children with clinically differentiated osteomyelitis exhibit distinct transcriptomic signatures.

There is substantial genomic heterogeneity among Staphylococcus aureus isolates of children with acute hematogenous osteomyelitis (AHO) but transcriptional behavior of clinically differentiated strains has not been previously described. This study evaluates transcriptional activity of S. aureus isolates of children with AHO that may regulate metabolism, biosynthesis, or virulence during bacterial growth and pathogenesis. In vitro growth kinetics were compared between three S. aureus clinical isolates from children with AHO who had mild, moderate, and severe illness. Total RNA sequencing was performed for each isolate at six separate time points throughout the logarithmic phase of growth. The NASA RNA-Sequencing Consensus Pipeline was used to identify differentially expressed genes allowing for 54 comparisons between the three isolates during growth. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment pathways were used to evaluate transcriptional variation in metabolism, biosynthesis pathways and virulence potential of the isolates. The S. aureus isolates demonstrated differing growth kinetics under standardized conditions with the mild isolate having higher optical densities with earlier and higher peak rates of growth than that of the other isolates (p<0.001). Enrichment pathway analysis established distinct transcriptional signatures according to both sampling time and clinical severity. Moderate and severe isolates demonstrated pathways of bacterial invasion, S. aureus infection, quorum sensing and two component systems. In comparison, the mild strain favored biosynthesis and metabolism. These findings suggest that transcriptional regulation during the growth of S. aureus may impact the pathogenetic mechanisms involved in the progression of severity of illness in childhood osteomyelitis. The clinical isolates studied demonstrated a tradeoff between growth and virulence. Further investigation is needed to evaluate these transcriptional pathways in an animal model or during active clinical infections of children with AHO.

Early Transition to Oral Antimicrobial Therapy Among Children With Staphylococcus aureus Bacteremia and Acute Hematogenous Osteomyelitis.

BACKGROUND: Staphylococcus aureus bacteremia (SAB) is a frequent complication of acute hematogenous osteomyelitis (AHO) in children, but data on the optimal duration of parenteral antibiotics prior to transition to oral antibiotics remains sparse. We examined clinical outcomes associated with early transition to oral antimicrobial therapy among children admitted to our institution with AHO and SAB, and evaluated the utility of a severity of illness score (SIS) to guide treatment decisions in this setting. METHODS: Children with AHO and SAB admitted to our institution between January 1, 2009, and December 31, 2018, were retrospectively reviewed and stratified according to a previously validated SIS into mild (0-3), moderate (4-7) and severe (8-10) cohorts. Groups were assessed for differences in treatment (eg, parenteral and oral antibiotic durations, surgeries) and clinical response (eg, bacteremia duration, acute kidney injury, length of stay and treatment failure). RESULTS: Among 246 children identified with AHO and SAB, median parenteral antibiotic duration differed significantly between mild (n = 80), moderate (n = 98) and severe (n = 68) cohorts (3.6 vs. 6.5 vs. 14.3 days; P ≤ 0.001). SIS cohorts also differed with regard to number of surgeries (0.4 vs. 1.0 vs. 2.1; P ≤ 0.001), duration of bacteremia (1.0 vs. 2.0 vs. 4.0 days; P ≤ 0.001), acute kidney injury (0.0% vs. 3.0% vs. 20.5%; P ≤ 0.001), hospital length of stay (4.8 vs. 7.4 vs. 16.4 days; P ≤ 0.001) and total duration of antibiotics (34.5 vs. 44.7 vs. 60.7 days; P ≤ 0.001). Early transition to oral antimicrobial therapy among mild or moderate SIS cohorts was not associated with treatment failure despite SAB. CONCLUSIONS: SAB is associated with a wide range of illness among children with AHO, and classification of severity may be useful for guiding treatment decisions. Early transition to oral antimicrobial therapy appears safe in children with mild or moderate AHO despite the presence of SAB.

Quality Improvement of Magnetic Resonance Imaging for Musculoskeletal Infection in Children Results in Decreased Scan Duration and Decreased Contrast Use.

BACKGROUND: Magnetic resonance imaging (MRI) is a heavily utilized resource to evaluate children suspected to have a musculoskeletal infection. Complex interdisciplinary workflows are involved with decision-making with regard to indications, anesthesia, contrast use, and procedural timing relative to the scan. This study assesses the impact of a quality improvement endeavor on MRI workflows at a tertiary pediatric medical center. METHODS: A registry of consecutively enrolled children for a multidisciplinary musculoskeletal infection program identified those evaluated with MRI from 2012 to 2018. Annual MRI process improvement feedback was provided to the key stakeholders. Demographic characteristics, laboratory parameters, MRI indications, anesthesia use, MRI findings, final diagnoses, scan duration, imaging protocol, surgical intervention following MRI, and length of stay were retrospectively compared between the 3 cohorts (initial, middle, and final) representing 2-year increments to assess the impact of the initiative. RESULTS: There were 526 original MRI scans performed to evaluate 1,845 children with suspected musculoskeletal infection. Anesthesia was used in 401 children (76.2%). When comparing the initial, middle, and final study period cohorts, significant improvement was demonstrated for the number of sequences per scan (7.5 sequences for the initial cohort, 5.8 sequences for the middle cohort, and 4.6 sequences for the final cohort; p < 0.00001), scan duration (73.6 minutes for the initial cohort, 52.1 minutes for the middle cohort, and 34.9 minutes for the final cohort; p < 0.00001), anesthesia duration (94.1 minutes for the initial cohort, 68.9 minutes for the middle cohort, and 53.2 minutes for the final cohort; p < 0.00001), and the rate of contrast use (87.6% for the initial cohort, 67.7% for the middle cohort, and 26.3% for the final cohort; p < 0.00001). There was also a trend toward a higher rate of procedures under continued anesthesia immediately following the MRI (70.2% in the initial cohort, 77.8% in the middle cohort, and 84.6% in the final cohort). During the final 6-month period, the mean scan duration was 24.4 minutes, anesthesia duration was 40.9 minutes, and the rate of contrast administration was 8.5%. CONCLUSIONS: Progressive quality improvement through collaborative interdisciplinary communication and workflow redesign led to improved utilization of MRI and minimized contrast use for suspected musculoskeletal infection. There was a high rate of procedural intervention under continued anesthesia for children with confirmed musculoskeletal infection. LEVEL OF EVIDENCE: Therapeutic Level IV. See Instructions for Authors for a complete description of levels of evidence.

Severity Adjusted Risk of Long-term Adverse Sequelae Among Children With Osteomyelitis.

BACKGROUND: The purpose of this investigation was to evaluate the risk for long-term, adverse outcomes among children with osteomyelitis. METHODS: Children with osteomyelitis were prospectively enrolled from 2012 to 2014. Care was accomplished by a multidisciplinary team according to an institutional algorithm. Data were collected to define the severity of illness during the initial hospitalization and assess short, intermediate and long-term outcomes. Clinical examination, radiographic assessment and functional outcome survey administration were performed at a minimum of 2 year follow-up. A comparison cohort analysis was performed according to initial severity of illness score of mild (0-2), moderate (3-6) and severe (7-10). RESULTS: Of 195 children enrolled, 139 (71.3%) returned for follow-up at an average of 2.4 years (range, 2.0-5.0 years). Children with severe illness were less likely to have normal radiographs (severe, 4.0%; moderate, 38.2%; mild, 53.2%, P < 0.0001), and more likely to have osteonecrosis, chondrolysis, or deformity (severe, 32.0%; moderate, 5.9%; mild, 1.3%, P < 0.0001). Functional outcome measures did not significantly differ between severity categories. By regression analysis severity of illness score, plus age less than 3 years and Methicillin-resistant Staphylococcus aureus predicted severe sequelae with an area under the curve of 0.8617 and an increasing odds ratio of 1.34 per point of increase in severity score. CONCLUSION: Long-term severe adverse outcomes among children with osteomyelitis occurred in 11 of 139 (7.9%) children and were predicted by initial severity of illness. Other risks that diminished the likelihood of complete resolution or increased the risk of severe sequelae included Methicillin-resistant Staphylcoccus aureus etiology and young age. The majority of children with osteomyelitis do not require long-term follow-up beyond the initial treatment period.

Differentiation of Deep Venous Thrombosis Among Children With or Without Osteomyelitis.

BACKGROUND: Children with osteomyelitis are at risk for deep venous thrombosis (DVT). This study evaluates the characteristics of DVT among children to differentiate between those with and without osteomyelitis. METHODS: Children with DVT of any cause were studied between 2008 and 2016. Children with DVT and osteomyelitis were compared with those with DVT without osteomyelitis. Another comparison cohort included children with osteomyelitis but without DVT. Comorbidities, severity of illness (SOI), and clinical course were compared between cohorts. RESULTS: DVT was identified in 224 children, a prevalence of 2.5 per 10,000 children. Among those with DVT, 28 (12.1%) had osteomyelitis. The DVT rate among 466 children with osteomyelitis was 6.0%. Children with osteomyelitis and DVT had greater SOI (9.1 vs. 2.7), bacteremia rate (82.1% vs. 38.4%), methicillin-resistant Staphylococcus aureus rate (89.3% vs. 21.2%), surgeries per child (2.1 vs. 0.7), and intensive care unit admission rate (67.9% vs. 5.9%) than that of children without DVT (P<0.00001). Of 196 children who had DVT without osteomyelitis, 166 (84.7%) had comorbidities including defined hypercoagulability (27 or 13.8%). Children with DVT due to osteomyelitis were without comorbidities or hypercoagulability (P<0.00001). The rate of pulmonary embolism was similar for children with DVT with or without osteomyelitis (3/28, or 10.7% vs. 18/196, or 9.2%). CONCLUSIONS: Children with DVT and osteomyelitis differ substantially from other children with DVT by the absence of comorbidities or post-thrombotic syndrome. They also differ from children with osteomyelitis without DVT by higher SOI, methicillin-resistant S. aureus rate, and occurrence of intensive care. Awareness of for the characteristics of DVT among children with osteomyelitis will reduce delay to diagnostic ultrasound and improve anticoagulation management which must be carefully coordinated given the high rate of surgery of these children. LEVEL OF EVIDENCE: Level II-prognostic, retrospective cohort comparison.

The impact of Staphylococcus aureus genomic variation on clinical phenotype of children with acute hematogenous osteomyelitis.

BACKGROUND: Children with acute hematogenous osteomyelitis (AHO) have a broad spectrum of illness ranging from mild to severe. The purpose of this study is to evaluate the impact of genomic variation of Staphylococcus aureus on clinical phenotype of affected children and determine which virulence genes correlate with severity of illness. METHODS: De novo whole genome sequencing was conducted for a strain of Community Acquired Methicillin Resistant Staphylococcus aureus (CA-MRSA), using PacBio Hierarchical Genome Assembly Process (HGAP) from 6 Single Molecule Real Time (SMRT) Cells, as a reference for DNA library assembly of 71 Staphylococcus aureus isolates from children with AHO. Virulence gene annotation was based on exhaustive literature review and genomic data in NCBI for Staphylococcus aureus. Clinical phenotype was assessed using a validated severity score. Kruskal-Wallis rank sum test determined association between clinical severity and virulence gene presence using False Discovery Rate (FDR), significance <0.01. RESULTS: PacBio produced an assembled genome of 2,898,306 bp and 2054 Open Reading Frames (ORFs). Annotation confirmed 201 virulence genes. Statistical analysis of gene presence by clinical severity found 40 genes significantly associated with severity of illness (FDR ≤0.009). MRSA isolates encoded a significantly greater number of virulence genes than did MSSA (p < 0.0001). Phylogenetic analysis by maximum likelihood (PAML) demonstrated the relatedness of genomic distance to clinical phenotype. CONCLUSIONS: The Staphylococcus aureus genome contains virulence genes which are significantly associated with severity of illness in children with osteomyelitis. This study introduces a novel reference strain and detailed annotation of Staphylococcus aureus virulence genes. While this study does not address bacterial gene expression, a platform is created for future transcriptome investigations to elucidate the complex mechanisms involved in childhood osteomyelitis.