Staphylococcus aureus causes a variety of infection entities ranging from skin and soft tissue infections (SSTIs) to invasive bloodstream infections, osteomyelitis and pneumonia. Asymptomatic S. aureus colonization is present in approximately one-third of the United States population and is a risk factor for infection.1 Furthermore, recurrent S. aureus SSTIs are problematic; following treatment, up to 60% of patients with S. aureus SSTI will experience recurrence over 12 months.2
The effectiveness of preventive and treatment approaches to this versatile pathogen is threatened by burgeoning bacterial resistance. Most notable is the emergence of methicillin-resistant S. aureus (MRSA) strains, but there is also increasing resistance to commonly prescribed decolonization agents, including mupirocin and chlorhexidine.3 Novel strategies for infection prevention are needed. Endogenous micronutrients, such as 25-hydroxyvitamin D [25(OH)D] or vitamin D, have potential roles as mediators of protective immunity. Though well known for its role in bone metabolism and calcium homeostasis, vitamin D has recently garnered attention for its influence on host immunity in infectious diseases and autoimmune conditions.4–7 The active form of vitamin D [1,25(OH)2 D3] regulates transcription of antimicrobial peptides (AMPs) in keratinocytes, macrophages and neutrophils.8 The human cathelicidin AMP, termed LL-37, promotes first-line defense mechanisms, including vitamin D3-induced autophagy in macrophages and in vitro killing of MRSA.9,10 Therefore, it is biologically plausible that vitamin D might aid in prevention of S. aureus SSTI.
The high prevalence of vitamin D deficiency (7.6 million US children and adolescents4) encourages further study of the antimicrobial benefits of this micronutrient. Using sera from pediatric patients enrolled in a previous observational study of host immune responses to staphylococcal toxins,11 we investigated the relationship between serum 25(OH)D concentrations and S. aureus clinical phenotypes—specifically primary SSTI, recurrent SSTI and invasive disease. Our principal hypothesis was that patients with recurrent SSTI would have lower serum 25(OH)D levels than patients presenting with primary SSTI.
PATIENTS AND METHODS
Subject Recruitment and Sample Collection
Healthy children older than 6 months of age (n = 202) were recruited from St. Louis Children’s Hospital between August 2008 and September 2011 as described previously.11 Three groups were evaluated: primary (first-time) SSTI, recurrent SSTI, and invasive disease (eg, bacteremia, osteomyelitis and septic arthritis).
Colonization cultures of the nares, axillae and inguinal folds were taken from all participants at study enrollment as previously described.11 An enrollment survey collected information regarding demographics, acute infection symptoms, past medical history, household factors and activities. Sera were drawn at the time of acute infection. Study procedures were approved by the Washington University Human Research Protection Office. Written informed consent (and assent when appropriate) was obtained for each participant.
Laboratory Testing and Vitamin D Status Definition
Serum 25(OH)D levels were measured on 300-μL samples via an electrochemiluminescence immunoassay on the Roche Elecsys 2010 analyzer (Roche Diagnostics, Indianapolis, IN), which has an analytic sensitivity of 5.0 ng/mL. Two levels of quality controls were within the specified acceptable range. Precision was reflected in between-run coefficients of variation of 10.2% at 7.48 ng/mL and 3.2% at 53.12 ng/mL and within-run coefficients of variation of 2.53% at 13.4 ng/mL and 3.27% at 29.21 ng/mL. Serum 25(OH)D levels were categorized as follows5,6: sufficient ≥30 ng/mL, insufficient 20–29 ng/mL, and deficient <20 ng/mL.
Statistical analyses utilized SPSS 22 for Windows (IBM SPSS, Chicago, IL). Pearson’s χ2 tests were used to evaluate association between 25(OH)D level and variables a priori thought to be associated with 25(OH)D level and/or S. aureus infection type. These analyses were performed twice, categorizing 25(OH)D levels using 2 approaches: (1) separating 25(OH)D levels into all 3 categories (sufficient, insufficient, and deficient) and (2) condensing 25(OH)D levels into 2 categories (sufficient and insufficient/deficient) based on existing literature.5 Two separate logistic regression analyses were run using the backwards stepwise method. The first regression sought to determine which variables were associated with the outcome 25(OH)D insufficiency/deficiency. Variables included in the multivariable regression model were those significantly associated with 25(OH)D insufficiency/deficiency in univariate analyses. The second regression determined variables associated with the outcome infection type. This model was run twice comparing different infection types: primary versus recurrent SSTI and primary SSTI versus invasive infection. Odds ratios (OR) and 95% confidence intervals (CI) are reported. All tests of significance were 2-tailed; p values <0.05 were considered significant.
The median age among 202 participants was 7.6 years (range, 0.6–22.5). The study population was 48% African American (n = 97) and 53% female (n = 107). Eighty-six (43%) participants experienced primary SSTI, 62 (31%) experienced recurrent SSTI and 54 (27%) had invasive S. aureus infection. When categorized by 25(OH)D levels, 74 (37%) participants were deficient, 70 (35%) were insufficient, and 58 (29%) were sufficient. The overall mean 25(OH)D level was 23.7 ± 10.2 ng/mL. Mean 25(OH)D levels for the primary SSTI, recurrent SSTI and invasive infection cohorts were 24.3 ± 9.9, 23.8 ± 8.9 and 22.6 ± 12.0 ng/mL, respectively.
Age, race and season of infection were significantly associated with 25(OH)D values in univariate analysis [see Table, Supplemental Digital Content 1, http://links.lww.com/INF/C75, which includes potential associated factors by categorical 25(OH)D serum levels]. Deficient 25(OH)D levels were detected most frequently in the 13–22 years (oldest) age group (63%), compared with 7–12 years of age (36%) and those <7 years of age (20%; p < 0.001). African Americans were more likely to have deficient 25(OH)D levels (56%) compared with Caucasian/other races (19%; p < 0.001). Lastly, participants with infections during fall and winter (October–March) were more likely to have deficient 25(OH)D levels (50%) compared with those with infections in spring and summer (April–September; 28%; p < 0.001). Other factors, such as gender, BMI, weight for age, antibiotic use in the past year, hospitalization in the past year, S. aureus colonization and type of infecting organism (MRSA vs. methicillin-susceptible S. aureus), showed no significant association with 25(OH)D level. Factors associated with vitamin D deficiency/insufficiency in multivariable analysis (including age, season, race and study arm/infection type) were older age (increase of 1 year associated with OR, 1.14; 95% CI, 1.07–1.21) and fall/winter season (OR, 2.89; 95% CI, 1.41–5.91) when compared with vitamin D sufficiency.
Factors associated with presentation with a recurrent S. aureus SSTI (compared with primary SSTI) in multivariable analysis (including age, season, race and vitamin D level) were insufficient/deficient 25(OH)D levels (OR, 2.30; 95% CI, 1.02–5.21) and younger age (increase of 1 year associated with OR, 0.91; 95% CI, 0.86–0.97). A similar multivariable regression analysis comparing groups of patients presenting with a first-time S. aureus infection (SSTI vs. invasive infection) did not demonstrate an association between vitamin D level and infection type.
Given the role of vitamin D in regulation of LL-37 expression and the bactericidal activity of LL-37 against S. aureus, we investigated serum vitamin D levels in patients with a range of S. aureus infections. The current observational study supports a plausible association between serum 25(OH)D levels and host susceptibility to S. aureus SSTIs. Consistent with other studies, African-American race was associated with hypovitaminosis D.4 The increased melanin content of African-American skin affects UV absorption, subsequently yielding lower vitamin D stores. Interestingly, Liu et al.12 also found racial differences in induction of cathelicidin mRNA, which may also impact racial susceptibility to infection. Infection in winter months was also associated with lower vitamin D levels, which may be explained by diminished sun exposure and greater time spent indoors.4
Several studies have investigated the relationship of serum vitamin D levels and asymptomatic S. aureus colonization. Matheson et al.6 determined that individuals with vitamin D deficiency had increased risk of MRSA nasal carriage. On the contrary, Slow et al. found no correlation between 25(OH)D and S. aureus carriage risk, and vitamin D3 supplementation did not reduce persistent nasal carriage.13 Upon adjusting for race, age and season of infection presentation, we found that children with deficient or insufficient vitamin D levels (<30 ng/mL) were at a greater risk for their SSTI to be recurrent rather than primary. Perhaps AMPs, which are regulated by vitamin D, play a larger role in preventing infection rather than inhibiting colonization.
Several limitations should be noted. First, this observational study is a secondary analysis of samples used in a previous study examining humoral responses to S. aureus infection. As a result, we lack data regarding potential confounders, such as patient sun exposure, diet and vitamin supplementation use. Second, our cross-sectional analysis precludes determination of which occurred first: onset of infection or nonsufficient vitamin D levels. Thus, only associative rather than causative relationships can be inferred.
In a collection of observational studies, vitamin D level has been shown to have a significant correlation with other clinical outcomes, including atopic dermatitis and acute otitis media.5,7 Vitamin D supplementation trials have been successful in reducing recurrent uncomplicated acute otitis media episodes and severity of eczema.5,7 With biological evidence showing vitamin D-dependent regulation of AMPs (eg, LL-37) during a cutaneous infection episode, vitamin D supplementation may potentially improve clinical outcomes of S. aureus patients.8,12 Because recurrent SSTIs pose a substantial burden and the prevalence of vitamin D deficiency and insufficiency is high, additional studies are needed to further support the immunoregulatory benefits of vitamin D. As vitamin D sufficiency may be one of a myriad of host and environmental factors influencing S. aureus SSTI, vitamin D supplementation may serve as a safe and economical approach to reduce recurrent S. aureus infections.
The authors thank the Core Laboratory for Clinical Studies [Richard E. Ostlund, MD and Licia R. Rowe, MT (ASCP)] for their help with the vitamin D electrochemiluminescence assay, as well as Michael Wallendorf, PhD for assisting with data analysis.
1. Kluytmans J, van Belkum A, Verbrugh H.. Nasal carriage of Staphylococcus aureus
: epidemiology, underlying mechanisms, and associated risks. Clin Microbiol Rev. 1997;10:505–520
2. Fritz SA, Hogan PG, Hayek G, et al. Household versus individual approaches to eradication of community-associated Staphylococcus aureus
in children: a randomized trial. Clin Infect Dis. 2012;54:743–751
3. Fritz SA, Hogan PG, Camins BC, et al. Mupirocin and chlorhexidine resistance in Staphylococcus aureus
in patients with community-onset skin and soft tissue infections. Antimicrob Agents Chemother. 2013;57:559–568
4. Kumar J, Muntner P, Kaskel FJ, et al. Prevalence and associations of 25-hydroxyvitamin D
deficiency in US children: NHANES 2001–2004. Pediatrics. 2009;124:e362–e370
5. Marchisio P, Consonni D, Baggi E, et al. Vitamin D
supplementation reduces the risk of acute otitis media in otitis-prone children. Pediatr Infect Dis J. 2013;32:1055–1060
6. Matheson EM, Mainous AG 3rd, Hueston WJ, et al. Vitamin D
and methicillin-resistant Staphylococcus aureus
nasal carriage. Scand J Infect Dis. 2010;42:455–460
7. Samochocki Z, Bogaczewicz J, Jeziorkowska R, et al. Vitamin D
effects in atopic dermatitis. J Am Acad Dermatol. 2013;69:238–244
8. Lowry MB, Guo C, Borregaard N, et al. Regulation of the human cathelicidin
antimicrobial peptide gene by 1α,25-dihydroxyvitamin D3 in primary immune cells. J Steroid Biochem Mol Biol. 2014;143:183–191
9. Travis SM, Anderson NN, Forsyth WR, et al. Bactericidal activity of mammalian cathelicidin
-derived peptides. Infect Immun. 2000;68:2748–2755
10. Yuk JM, Shin DM, Lee HM, et al. Vitamin D3 induces autophagy in human monocytes/macrophages via cathelicidin
. Cell Host Microbe. 2009;6:231–243
11. Fritz SA, Tiemann KM, Hogan PG, et al. A serologic correlate of protective immunity against community-onset Staphylococcus aureus
infection. Clin Infect Dis. 2013;56:1554–1561
12. Liu PT, Stenger S, Li H, et al. Toll-like receptor triggering of a vitamin D
-mediated human antimicrobial response. Science. 2006;311:1770–1773
13. Slow S, Priest PC, Chambers ST, et al. Effect of vitamin D3 supplementation on Staphylococcus aureus
nasal carriage; a randomized, double-blind, placebo-controlled trial in healthy adults. Clin Microbiol Infect. 2014;20:453–458