Volume 13, Issue 6 , Pages e437-e443, November 2009
Staphylococcus aureus nasal carriage might be associated with vitamin D receptor polymorphisms in type 1 diabetes
Article Outline
Summary
Background
Polymorphisms in the vitamin D receptor (VDR) gene have been associated with susceptibility to several diseases, including type 1 diabetes (T1D) and infections. In this study we investigated whether VDR gene polymorphisms influence nasal carriage of Staphylococcus aureus in individuals with T1D.
Methods
In 93 T1D patients, VDR polymorphisms on FokI F>f, BsmI B>b, ApaI A>a, and TaqI T>t were determined in DNA extracted from peripheral blood leukocytes, and a nasal swab was obtained to detect colonization by S. aureus. A repeat swab was obtained in 76/93 subjects for the estimation of persistent S. aureus carriage.
Results
The prevalence of S. aureus nasal colonization was 31.2% and the prevalence of persistent carriage was 25%. The presence of TaqI T allele was related to higher rates of S. aureus colonization, and TaqI TT homozygotes were more colonized (48.5% vs. 21.7%; p 0.007; OR 3.40, 95% CI 1.36–8.52) and more persistent carriers (37.9% vs. 17.0%; p 0.039; OR 2.98, 95% CI 1.02–8.67). The presence of ApaI A allele was related to lower rates of S. aureus colonization, and ApaI AA homozygotes were less colonized (17.6% vs. 39.0%; p 0.026; OR 0.34, 95% CI 0.12–0.94) and less persistent carriers (11.5% vs. 32%; p 0.043; OR 0.28, 95% CI 0.07–1.06). No differences were observed for BsmI and FokI genotypes.
Conclusions
Our findings suggest that VDR polymorphisms may be associated with nasal carriage of S. aureus in individuals with T1D, and further contribute to the better understanding of the immunomodulatory role of vitamin D in the human host's response and susceptibility to infection.
Keywords: Vitamin D receptor polymorphisms, Type 1 diabetes, Staphylococcus aureus colonization, Staphylococcus aureus carriage, Susceptibility to infection
Introduction
The vitamin D endocrine system has pleiotropic effects on various processes including calcium absorption and bone biology, cell growth and differentiation, and modulation of the immune system.1, 2 Vitamin D mediates its actions by binding to the vitamin D receptor (VDR), which is a soluble protein present in various tissues and in T and B lymphocytes, monocytes, and macrophages.1, 2, 3, 4 Polymorphisms in the human VDR gene have been associated with several diseases including type 1 diabetes (T1D),2, 5, 6, 7 and with susceptibility to several bacterial and viral infections.3, 4, 8, 9, 10, 11, 12
Staphylococcus aureus is a major pathogen for humans with increasing resistance to antibiotics. The anterior nares are the primary ecological reservoir of S. aureus, and nasal carriage has been related to progress to staphylococcal disease.13, 14, 15, 16, 17, 18, 19, 20 The host's response seems to be crucial in S. aureus nasal colonization and carriage, but little is known in this field.20 Vitamin D metabolites are known to affect macrophage activation, and neutrophils have been demonstrated to express functional VDR,10, 21 hence VDR gene polymorphisms may affect colonization and infection by microorganisms such as S. aureus that might behave as an intracellular pathogen.
Individuals with T1D are considered to be susceptible to infections in terms of frequency, severity, and complications, including infections by S. aureus.13, 15, 16, 22 Although VDR polymorphisms are related both with T1D and susceptibility to infection, and T1D is further associated to S. aureus infection, to date no study has investigated potential links between VDR polymorphisms on the one hand and T1D and S. aureus disease or carriage on the other. In this study we investigated whether the four most commonly studied polymorphisms in the VDR gene, i.e., FokI F>f, BsmI B>b, ApaI A>a, and TaqI T>t are associated with colonization by and persistent carriage of S. aureus in individuals with T1D.
Patients and methods
Patient recruitment
The study group consisted of a cohort of 93 T1D patients, followed up at the outpatient diabetic clinic of the Department of Pediatrics, University Hospital of Heraklion, Crete, Greece. This T1D outpatient clinic is the only one on the island of Crete. Diagnosis of T1D follows the American Diabetes Association guidelines.23 The study protocol was approved by the institutional committees of the University Hospital of Heraklion and the Medical School, University of Crete.
S. aureus carriage
Nasal specimens were obtained by rotating a sterile fiber-tipped swab four times in both anterior nares of each enrolled individual. The swabs were immediately placed in Amies transport medium (BioMerieux, Marcy L’Etoile, France) and kept at 4
°C before being inoculated onto mannitol salt agar and Columbia agar with 5% sheep blood. The culture plates were incubated at 36°C for 48hours. S. aureus was identified on the basis of colony morphology, Gram stain, catalase and coagulase test, and the API 20 Staph system (BioMerieux). A second nasal swab culture was obtained after a minimum interval of 3 months. Subjects were classified as persistent carriers if both cultures were positive, as non-carriers if both cultures were negative, and as intermittent carriers in cases where one culture was positive and the other was negative.13, 15, 17, 18, 19, 20, 24 Individuals with a single nasal swab available were excluded from further analysis of persistent carriage.
DNA analysis
Whole blood was collected in EDTA-containing tubes, and genomic DNA was isolated from peripheral blood leukocytes using the Puregene kit (Gentra Systems, Minnesota, USA). The extracted DNA was stored at −20°C until analysis. DNA was amplified with standard PCR techniques. The cDNA of the human VDR (vitamin D 1,25-dihydroxyvitamin D3 receptor) sequence (GenBank accession number J03258) was used to design primers for PCR amplification of the fragments, as shown in Table 1. Both undigested and digested PCR products were visualized on 2.5% agarose gels stained with ethidium bromide. Genotypes were determined according to the presence or absence of an appropriate restriction site, and alleles were designated respective to actual base change according to the dbSNP (http://www.ncbi.nlm.nih.gov/projects/SNP/). Genotypes were scored blindly and analysis of all ambiguous samples was repeated. Moreover, 10% of the samples were amplified twice to check the accuracy of results. The selection of the four enzyme sites followed previous protocols, and the established nomenclature for restriction fragment length polymorphism alleles was used in this study.2, 7, 10, 11, 12, 18 The lowercase allele represents the presence of the restriction site (f: FokI, b: BsmI, a: ApaI, or t: TaqI) and the uppercase allele represents the absence of the restriction site (F, B, A, or T, respectively).25 Allele types, SNP reference numbers, and PCR conditions for all the analyzed polymorphisms are shown in Table 2.
Table 1. PCR primers designed to amplify fragments harboring the vitamin D receptor single nucleotide polymorphisms (SNPs)
| SNP | PCR primer | Fragment size |
|---|---|---|
| ApaI | Forward (5′–3′) CAG AGC ATG GAC AGG GAG CAA | 740 bp uncleaved |
| Reverse (3′–5′) GCA ACT CCT CAT GGC TGA GGT C CTC | 530 bp, 210 bp | |
| BsmI | Forward (5′–3′) CAA CCA AGA CTA CAA GTA CCG CGT CAG TGA | 825 bp uncleaved |
| Reverse (3′–5′) AAC CAG CGG GAA GAG GTC AAG GG | 650 bp, 175 bp | |
| FokI | Forward (5′–3′) AGC TGG CCC TGG CAC TGA CTC TGC TCT | 265 bp uncleaved |
| Reverse (3′–5′) ATG GAA ACA CCT TGC TTC TTC TCC CTC | 196 bp, 69 bp | |
| TaqI | Forward (5′–3′) CAG AGC ATG GAC AGG GAG CAA | 740 bp uncleaved |
| Reverse (3′–5′) GCA ACT CCT CAT GGC TGA GGT C CTC | 495 bp, 245 bp or 290 bp, 245 bp and 205 bp |
Table 2. Allele types, single nucleotide polymorphism (SNP) reference numbers, and PCR conditions
| SNP | Allele type | Ref. number | PCR conditions |
|---|---|---|---|
| ApaI | a allele: T→G transition in intron 8 | rs7975232 | Denaturation at 94°C for 5min, followed by addition of the polymerase, and then samples were exposed to 35 cycles of denaturing (at 94°C for 30 s), annealing (at 70°C for 30 s), and chain extension (at 72°C for 1 min), followed by a final extension step at 72°C for 7 min |
| BsmI | b allele: G→A transition | rs1544410 | Initial heating at 94°C for 5min, followed by addition of the polymerase and then 35 cycles of denaturing (at 94°C for 15 s), annealing (at 60°C for 30 s), and chain extension (at 72°C for 30 s), followed by a final extension step at 72°C for 7 min |
| FokI | f allele: C→T transition at the junction of intron 1 and exon 2 | rs10735810 | Initial heating at 94°C for 5min, followed by 35 cycles of denaturing (at 94°C for 15 s), annealing (at 60°C for 30 s), and chain extension (at 72°C for 30 s), followed by a final extension step at 72°C for 7 min |
| TaqI | t allele: silent T→C transition in exon 9 | rs731236 | Denaturation at 94°C for 5min, followed by addition of the polymerase, and then samples were exposed to 35 cycles of denaturing (at 94°C for 30 s), annealing (at 70°C for 30 s), and chain extension (at 72°C for 1 min), followed by a final extension step at 72°C for 7 min |
Confirmation of polymorphisms by direct sequencing
Selected PCR amplified fragments from the four VDR examined polymorphisms, corresponding to heterozygous or mutant homozygous genotypes, were completely sequenced, both strands, in a LiCor 4200L sequencer at the laboratory of microchemistry (IMBB-FORTH, Crete, Greece) in order to confirm that the amplified products represent genuine VDR regions.
Statistical analysis
Statistical analysis was based on contingency tables, including calculations of odds ratio (OR) and of the lower and upper limits of the 95% confidence interval (95% CI). The one-tailed Fisher's exact test was applied to determine whether distributions of categorized variables between the groups were significant. The Hardy–Weinberg equilibrium was tested by comparing expected and observed genotype frequencies by Chi-square test. The conventional level of p<0.05 was considered to be significant.
Results
During the 21-year period, 1986–2006, 147 individuals were diagnosed with T1D and followed-up at the outpatient clinic. Of these, 93 (63.3%) were enrolled in this study. The 93 individuals (43 female and 50 male) were aged 3.3 to 25.3 (mean 12.3, median 12.4) years, and the duration of T1D from diagnosis ranged from 0.1 to 17.0 (mean 4.4, median 2.8) years.
VDR gene FokI, BsmI, ApaI, and TaqI genotypes and allele frequencies are shown in Table 3. In the initial nasal swab sampling, 29/93 individuals (31.2%) were found to be colonized by S. aureus. Among the 76 individuals from whom a second culture was obtained, 19 (25%) were defined as persistent S. aureus carriers, 21 (27.6%) as intermittent carriers, and 36 (47.4%) as non-carriers for S. aureus.
Table 3. Distribution of VDR genotype and haplotype frequencies in 93 individuals with type 1 diabetes as related to S. aureus nasal colonization
| VDR gene polymorphism | HWEap-value | S. aureus colonized (%) | S. aureus non-colonized (%) | Total | p-Value; OR (95% CI)b |
|---|---|---|---|---|---|
| FokI | |||||
| Genotype | 0.67 (0.91) | NS | |||
| ff | 0 (0) | 7 (100) | 7 | ||
| Ff | 16 (40) | 24 (60) | 40 | ||
| FF | 13 (28.3) | 33 (71.7) | 46 | ||
| Allele | NS | ||||
| f | 16 (29.6) | 38 (70.4) | 54 | ||
| F | 42 (31.8) | 90 (68.2) | 132 | ||
| BsmI | |||||
| Genotype | 0.12 (0.30) | NS | |||
| bb | 6 (31.6) | 13 (68.4) | 19 | ||
| Bb | 19 (35.2) | 35 (64.8) | 54 | ||
| BB | 4 (20) | 16 (80) | 20 | ||
| Allele | NS | ||||
| b | 31 (33.7) | 61 (66.3) | 92 | ||
| B | 27 (28.7) | 67 (71.3) | 94 | ||
| ApaI | |||||
| Genotype | 0.01 (0.05) | 0.026; 0.34 (0.12–0.94)c | |||
| aa | 2 (33.3) | 4 (66.7) | 6 | ||
| Aa | 21 (39.6) | 32 (60.4) | 53 | ||
| AA | 6 (17.6) | 28 (82.4) | 34 | ||
| Allele | 0.081; 0.60 (0.31–1.14)d | ||||
| a | 25 (38.5) | 40 (61.5) | 65 | ||
| A | 33 (27.3) | 88 (72.7) | 121 | ||
| TaqI | |||||
| Genotype | 0.02 (0.07) | 0.007; 3.40 (1.36–8.52)e | |||
| tt | 1 (14.3) | 6 (85.7) | 7 | ||
| Tt | 12 (22.6) | 41 (77.4) | 53 | ||
| TT | 16 (48.5) | 17 (51.5) | 33 | ||
| Allele | 0.016; 2.22 (1.11–4.46)f | ||||
| t | 14 (20.9) | 53 (79.1) | 67 | ||
| T | 44 (37.0) | 75 (63.0) | 119 | ||
| Total patients | 29 (31.2) | 64 (68.8) | 93 | ||
| Total alleles | 58 (31.2) | 128 (68.8) | 186 | ||
aHardy–Weinberg equilibrium (calculations with 1 degree of freedom; in parentheses values after calculation with 2 degrees of freedom). |
bp-Values and odds ratios calculated for colonized vs. non-colonized T1D individuals. |
c(ApaI aa |
dA allele vs. a allele. |
eTaqI TT vs. (TaqI tt |
fT allele vs. t allele. |
VDR polymorphisms and initial S. aureus carriage
As shown in Table 3, S. aureus nasal colonization was more common in individuals with TaqI T than t allele (37.0% vs. 20.9%; p 0.016; OR 2.22, 95% CI 1.11–4.46), and TaqI TT homozygotes were more colonized by S. aureus than genotypes TaqI tt and Tt combined (48.5% vs. 21.7%; p 0.007; OR 3.40, 95% CI 1.36–8.52). S. aureus nasal colonization was less common in individuals with ApaI A rather than a allele, although not at a statistically significant level (27.3% vs. 38.5%; p 0.081; OR 0.60, 95% CI 0.31–1.14); furthermore ApaI AA homozygotes were less colonized than ApaI aa and Aa genotypes combined (17.6% vs. 39.0%; p 0.026; OR 0.34, 95% CI 0.12–0.94). No significant differences were observed for BsmI and FokI genotypes.
VDR polymorphisms and persistent S. aureus carriage
As shown in Table 4, S. aureus persistent nasal colonization was more common in individuals with TaqI T than t allele (29.3% vs. 17.0%), although at a marginally significant level (p 0.068; OR 2.03, 95% CI 0.88–4.68); furthermore TaqI TT homozygotes were persistently colonized by S. aureus more than genotypes TaqI tt and Tt combined (37.9% vs. 17.0%; p 0.039; OR 2.98, 95% CI 1.02–8.67). S. aureus nasal colonization was more common in individuals with ApaI a than A allele, although not at a significant level (21.9% vs. 30.4%; p 0.17; OR 0.64, 95% CI 0.30–1.36); furthermore, ApaI AA homozygotes were less persistent carriers than ApaI aa and Aa genotypes combined (11.5% vs. 32%; p 0.043; OR 0.28, 95% CI 0.07–1.06). No significant differences were observed for BsmI and FokI genotypes.
Table 4. Distribution of VDR genotype and haplotype frequencies in 76 individuals with type 1 diabetes as related to S. aureus persistent nasal carriage
| VDR gene polymorphism | HWEap-value | Persistent carriers (%) | Intermittent carriers (%) | Non-carriers (%) | Total | p-Value; OR (95% CI)b |
|---|---|---|---|---|---|---|
| FokI | ||||||
| Genotype | 0.98 (1.00) | NS | ||||
| ff | 0 (0.0) | 1 (14.3) | 6 (85.7) | 7 | ||
| Ff | 11 (34.4) | 9 (28.1) | 12 (37.5) | 32 | ||
| FF | 8 (21.6) | 11 (29.7) | 18 (48.7) | 37 | ||
| Allele | NS | |||||
| f | 11 (23.9) | 11 (23.9) | 24 (52.2) | 46 | ||
| F | 27 (25.5) | 31 (29.2) | 48 (45.3) | 106 | ||
| BsmI | ||||||
| Genotype | 0.07 (0.19) | NS | ||||
| bb | 4 (26.7) | 3 (20.0) | 8 (53.3) | 15 | ||
| Bb | 13 (28.3) | 13 (28.3) | 20 (44.4) | 46 | ||
| BB | 2 (13.3) | 5 (33.3) | 8 (53.4) | 15 | ||
| Allele | NS | |||||
| b | 21 (27.6) | 19 (25.0) | 36 (47.4) | 76 | ||
| B | 17 (22.4) | 23 (30.3) | 36 (47.3) | 76 | ||
| ApaI | ||||||
| Genotype | 0.03 (0.10) | 0.043; 0.28 (0.07–1.06)c | ||||
| aa | 1 (16.7) | 3 (50.0) | 2 (33.3) | 6 | ||
| Aa | 15 (34.1) | 8 (18.2) | 21 (47.7) | 44 | ||
| AA | 3 (11.5) | 10 (38.5) | 13 (50.0) | 26 | ||
| Allele | 0.17; 0.64 (0.30–1.36)d | |||||
| a | 17 (30.4) | 14 (25.0) | 25 (44.6) | 56 | ||
| A | 21 (21.9) | 28 (29.2) | 47 (48.9) | 96 | ||
| TaqI | ||||||
| Genotype | 0.10 (0.26) | 0.039; 2.98 (1.02–8.67)e | ||||
| tt | 1 (16.7) | 2 (33.3) | 3 (50.0) | 6 | ||
| Tt | 7 (17.1) | 14 (34.1) | 20 (48.8) | 41 | ||
| TT | 11 (37.9) | 5 (17.2) | 13 (44.8) | 29 | ||
| Allele | 0.068; 2.03 (0.88–4.68)f | |||||
| t | 9 (17.0) | 18 (34.0) | 26 (49.0) | 53 | ||
| T | 29 (29.3) | 24 (24.2) | 46 (46,5) | 99 | ||
| Total patients | 19 (25.0) | 21 (27.6) | 36 (47.4) | 76 | ||
| Total alleles | 38 (25.0) | 42 (27.6) | 72 (47.4) | 152 | ||
aHardy–Weinberg equilibrium (calculations with 1 degree of freedom; in parentheses values after calculation with 2 degrees of freedom). |
bp-Values and odds ratios calculated for persistent vs. intermittent plus non-carriage state. |
c(ApaI aa |
dA allele vs. a allele. |
eTaqI TT vs. (TaqI tt |
fT allele vs. t allele. |
Discussion
Our findings suggest that two VDR polymorphisms, both located in the 3′ region of the VDR gene, are related to colonization by and persistent nasal carriage of S. aureus in individuals with T1D. In this study we focused on both initial colonization and persistent carriage, as persistent carriers have higher S. aureus loads and a higher risk of acquiring S. aureus infection.13 The rates of colonization and persistent S. aureus carriage in the study population (31% and 25%, respectively) are consistent with the ranges reported elsewhere.15, 16, 17, 18, 19, 26
The ability of S. aureus to colonize the human host and progress to infection depends both on virulence properties of the invading strain and on the individual host's response. Not much is known on the intrinsic properties of S. aureus strains that facilitate colonization and long-term carriage.18, 19, 20 By contrast, evidence is emerging and pointing to host genetic factors affecting S. aureus colonization and carriage.18, 20, 24, 26 Staphylococcal carriage has long been associated with the presence of HLA-DR3.27 In individuals with T1D, dendritic cells have been shown to produce less interferon-α,28 and polymorphonuclear cells have demonstrated impaired killing function against S. aureus.16 Our findings provide evidence for involvement of the vitamin D immunomodulatory system. Interestingly, no significant association between VDR gene polymorphism and S. aureus nasal carriage status was demonstrated in a study with healthy volunteers.17 Hence, we assume that it is in conditions with impaired immunity, such as in T1D, where VDR polymorphisms may affect susceptibility to S. aureus colonization and carriage. VDR gene polymorphisms may be involved in colonization by S. aureus, since vitamin D metabolites have been shown to play a role in macrophage activation and differentiation10, 11, 21 and neutrophils have been demonstrated to play an important role in the innate immunity against S. aureus. Cole et al.18 demonstrated that persistent S. aureus nasal carriage is accompanied by the release of epithelial- and neutrophil-derived host defense peptides into nasal secretions, providing a possible explanation for the failure of S. aureus clearance in individuals with macrophage dysfunction.
The ApaI genotype has been shown to modulate susceptibility to Mycobacterium malmoense3 and hepatitis B virus,4 and the TaqI genotype susceptibility to mycobacteria,3, 9 lower respiratory tract infections,10 and periodontitis.11, 12 Vitamin D metabolites are known to suppress tumor necrosis factor-α, interleukin (IL)-1, and IL-2, to stimulate transforming growth factor-β1 and IL-4 production, to inhibit T cell proliferation and inflammatory activity,1 and thus to modulate Th1/Th2 balance,3 to modulate expression of neutrophil genes, and restrict association and invasion of macrophages by pathogens including S. aureus.20, 29 S. aureus carriage is associated with opsonization,13, 14 and persistent S. aureus nasal carriage has been shown to be accompanied by the release of epithelial- and neutrophil-derived peptides into nasal secretions.18 The TaqI polymorphism has been proposed to affect VDR-mediated production of IL-1α and IL-6 and production of anti-proteinase TIMP-1, which is a natural inhibitor of metalloproteinase-9.10 On the other hand, VDR gene polymorphisms FokI, BsmI, ApaI, and TaqI are well known to influence the occurrence of T1D, although protective or predisposing effects vary greatly among studies and populations worldwide.5, 6, 7
Our findings are based on a relatively small population and need to be confirmed in larger cohorts. However, as we enrolled the majority of T1D individuals in the island of Crete, our study provides the benefits of a well-defined area and of a population with one of the lowest T1D incidence rates in Europe.30 Violation of Hardy–Weinberg equilibrium in ApaI and TaqI polymorphisms in our study should be attributed to the small cohort size, and to inbreeding, a minimized migration rate, and closed gene pool of our island population. In addition, following suggestions by Chen and Chatterjee,31 we used a 2-degrees-of-freedom Chi-square ‘model-free’ test of association that has robust power under unknown modes of genetic effects, and confirmed that Hardy–Weinberg equilibrium was not violated any more (Table 3, Table 4). Violation of Hardy–Weinberg equilibrium has been observed in several other studies investigating VDR polymorphisms in T1D and has been attributed to disease-related gene, non-random mating, and common genetic background of study populations.32, 33, 34
As polymorphisms ApaI and TaqI are probably non-functional and do not modify the VDR polypeptide sequence, linkage disequilibrium with one or more truly functional polymorphisms elsewhere in the VDR gene is assumed to explain the impact on VDR functionality. Research for additional polymorphisms across the VDR gene is therefore required to verify this hypothesis. In a sequence analysis performed in our laboratory using Genomatix (Genomatix Software GmbH 1998–2008), the polymorphic site was not found to disrupt any transcription factor binding site and, therefore, it does not seem plausible that the SNPs under study affect the expression levels of VDR. Our study was only of a descriptive nature, and future research should include functional tests on immune cells and focus on underlying mechanisms and on genetic and environmental factors potentially interfering in the relationship between VDR polymorphisms and S. aureus carriage and disease. Furthermore, future research might use peripheral blood mononuclear cells instead of whole blood.
Our findings suggest that genetic variation in the vitamin D receptor gene affects nasal colonization by S. aureus in individuals with type 1 diabetes, and further contribute to the understanding of the immunoregulatory role of vitamin D in the host's response and susceptibility to infection.
Acknowledgements
We thank Professor Dimitrios T. Boumpas, Laboratory of Internal Medicine, University of Crete, for his contribution to this study.
Conflict of interest: No conflict of interest to declare.
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PII: S1201-9712(09)00119-2
doi:10.1016/j.ijid.2009.02.012
© 2009 International Society for Infectious Diseases. Published by Elsevier Inc. All rights reserved.
Volume 13, Issue 6 , Pages e437-e443, November 2009
