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Abstract

Aims/Introduction

Vitamin D deficiency is prevalent among individuals with diabetic foot infections (DFIs) and can influence a range of patient-related parameters. Hence, we aimed to find the association of serum vitamin D levels with various clinical, biochemical, and inflammatory parameters, as well as the microbial environment in the wounds of DFI patients.

Materials and Methods

From September 2022 to March 2024, 169 DFI patients participated in cross-sectional research at the hospital. Utilising an electrochemiluminescence immunoassay, vitamin D levels were determined. Vitamin D receptor (VDR) and Cathelicidin (LL-37) were estimated using ELISA Kits. The tissue culture plate technique was used to analyze biofilm formation. Patient-related parameters were obtained from medical records.

Results

The vitamin D status of DFI patients indicated that 70.4% were deficient, 19.5% were insufficient, and 10.1% were sufficient. The median vitamin D, VDR protein, and LL-37 values were 15.3 ng/mL, 0.832 ng/mL, and 1.54 ng/mL, respectively. Biofilm formation was seen in 84.36% of bacteria from vitamin D-deficient DFI patients (P < .001). Vitamin D levels were negatively correlated with ulcer duration, hospital stay, white blood cell count, neutrophil-lymphocyte ratio, C-reactive protein, and systemic inflammation index (r = −0.787, −0.849, −0.6, −0.475, −0.402, and −0.521, respectively; P < .001). However, vitamin D levels were positively correlated with VDR and LL-37 levels (r = 0.988 and 0.944, respectively; P < .001).

Conclusions

The majority of DFI patients exhibited vitamin D deficiency, which was significantly associated with most of the clinical, biochemical, and inflammatory parameters, in addition to the microbial environment within the wound.

Keywords

biofilm diabetic foot infection LL-37 vitamin D vitamin D receptor

Introduction

The International Diabetes Federation reports that diabetes mellitus (DM) affects approximately 10% of adults worldwide, equivalent to 589 million adults aged 20-79 years.¹ Projections predict an increase to 853 million by 2050.¹ A recent study estimates that 101 million people in India, or 11.4% of the country's population, currently have DM.² Various complications can arise in DM patients with uncontrolled blood sugar levels, with diabetic foot ulceration being one of the most critical.³ These ulcers are prone to the development of diabetic foot infections (DFIs), which may result in hospitalisation, lower limb amputation, disability, or even death.⁴ Approximately 10%-15% of patients with DFIs require multidisciplinary treatment. Of these cases, 20% classified as moderate or severe may necessitate amputation.⁵ Patients with DFIs face a 2.5-fold increase in mortality risk.⁵ In 2017, a survey indicated that the cost of managing DFIs in India was approximately 1572 USD per patient. Data shows that patients with DFIs incur expenses four times greater than those without DFIs.⁶

Several patient-related parameters can influence the development and severity of DFIs. These include age, gender, dietary pattern, the duration of diabetes, glycated haemoglobin (HbA1c), C-reactive protein (CRP), neutrophil-to-lymphocyte ratio (NLR), and systemic immune-inflammation index (SII).^7–13 Furthermore, hyperglycaemic state in these patients generates oxidative stress and sets off a chain of unfavourable consequences directly related to the deteriorating DFI prognosis.³ Among these consequences are a compromised immune system, chronic inflammation, microvascular dysfunction, challenges in wound healing, and the formation of biofilms.^3,14

Vitamin D, initially employed for treating nutritional rickets, is now widely used because of its numerous physiological benefits.^15,16 It functions by binding to the vitamin D receptor (VDR) found in skin and immune cells.¹⁷ Additionally, vitamin D works as an antibiotic by aiding the formation of antimicrobial peptides (AMPs) such as defensins and cathelicidin (LL-37) in different species.^18,19 In humans, LL-37 is a small AMP that demonstrates proangiogenic and immunomodulatory properties. Consequently, it has the capacity to improve the recovery process of wounds.²⁰ Vitamin D is one of the factors that regulate VDR and LL-37 expression.^21,22 Therefore, any disruptions in maintaining optimal vitamin D range could potentially impact the production of VDR and LL-37 and subsequently influence innate immunity and thereby affect wound healing.^23,24 Moreover, the growing challenge of treating patients with DFI is partly due to the existence of bacteria that create biofilms.²⁵ Furthermore, research demonstrates that a deficiency in vitamin D correlates with elevated levels of inflammatory cytokines, reduced LL-37, and the development of biofilms.^25–27 Evidence is also available on the vitamin D-LL-37 pathway's role in countering microbial protein.²⁸ Therefore, individuals with diabetes who have vitamin D deficiency are more susceptible to chronic and recurring DFI.^29,30 Also, vitamin D levels are associated with low-cost and widely accessible inflammatory markers, including white blood cell (WBC), CRP, NLR, and SII, indicating that vitamin D may have a part in controlling inflammation.^31–33

Considering the aforementioned evidence, this study aimed to estimate serum vitamin D levels in DFI patients and to determine its relationship with various clinical (age, duration of diabetes, specifics regarding their current ulcer, and hospital stays), biochemical (HbA1c, VDR protein and LL-37 levels), and inflammatory (WBC, CRP, NLR, and SII,) parameters as well as the microbial environment (biofilm) present in the wound.

Methods

Study Design and Ethical Approval

A cross-sectional study was carried out with 169 DFI patients who were admitted from September 2022 to March 2024 in the Department of General Surgery of the tertiary healthcare hospital. The institutional ethics committee (IEC) of the hospital has granted approval for the study, and the Helsinki Declaration (2013) ethical principles for human biomedical research were followed. The study documentation followed the STROBE guidelines.³⁴

Patient Enrolment

Patients with diabetes, both male and female, aged 18 and older, diagnosed with DFI according to the IWGDF/IDSA infection grading system, and those who provided consent were included.³⁵ The exclusion criteria encompassed patients with hepatic and renal disorders (which affect vitamin D metabolism), coeliac disease, chronic pancreatitis, Crohn's disease, cystic fibrosis (which impacts vitamin D absorption), and peripheral vascular disease. In addition, those who received immunosuppressants, chemotherapeutic agents, or corticosteroids within the preceding 30 days, and pregnant or lactating women were also excluded.^36,37 Moreover, patients on vitamin D supplements or any specific diet (fortified with vitamin D) and who did not consent to participate were excluded from the study. Additionally, patients with DFI whose wound samples indicated negligible bacterial growth were also excluded from the study.³⁸

In this study, a total of 424 patients were screened for eligibility. Among these patients, 154 had peripheral vascular disease, 21 had renal disorders, 9 had hepatic disorders, 57 had wound cultures that demonstrated negligible bacterial growth, and 5 patients did not give consent. As a result, all of these patients were excluded, leaving 178 who met the inclusion criteria. Furthermore, we excluded 9 patients because the preserved organisms from their wound samples displayed non-significant growth when regrown for biofilm analysis. Consequently, the final analysis included data from 169 patients with DFI. A flowchart illustrating the patient selection procedure is presented in Figure 1.

Figure 1.

Study Flowchart for the Selection of DFI Patients.

Data Collection Methods

Most of the data pertaining to patient-related parameters such as age, duration of diabetes, specifics regarding their current ulcer, hospital stays due to DFI, HbA1c levels, WBC count, and CRP levels were collected from the medical records. Dividing neutrophils by lymphocytes from the differential blood cell counts test yielded the NLR values. SII value = (Platelet × Neutrophil) / Lymphocyte count.

Measurement of Serum Vitamin D Levels in DFI Patients

Electrochemiluminescence immunoassay in the COBAS 6000 analyser was used to measure vitamin D levels in 4 mL blood samples from DFI patients at the hospital. Patients with vitamin D levels below 20 ng/mL were deficient, those between 20 and 30 were insufficient, and those over 30 were sufficient.³⁹

Estimation of Vitamin D Receptor and LL-37 Levels in Serum Samples of DFI Patients

4 mL of blood was taken from DFI patients utilising vacutainers to estimate the levels of VDR and LL-37 using the ELISA technique. The blood sample was centrifuged at 3000 rpm for 15 min to separate serum, which was stored at −80 °C until further analysis. The human VDR kit from Elabscience, USA (catalog number E-EL-H2043) was utilised for VDR estimation.⁴⁰ For LL-37, the estimation was performed using the antibacterial protein LL-37 kit from Elabscience, USA (catalog number E-EL-H2438).⁴¹ The procedures for estimating both VDR and LL-37 were conducted following the manufacturer's specifications.

Analysis of Biofilm Formation in Bacterial Isolates from Wound Samples of DFI Patients

200 bacterial isolates collected from wound samples of DFI patients were subjected to biofilm production test at the Department of Microbiology of the hospital. A matrix-assisted laser desorption ionisation time-of-flight (MALDI-TOF) - VITEK MS (BioMerieux, Marcy l’Etoile) system was used to identify bacterial isolates from the wounds of DFI patients. Subsequently, the VITEK 2 system (BioMérieux, Inc., Durham, NC) was used to test the samples for their antimicrobial susceptibility.⁴²

The tissue culture plate method was employed to assess the biofilm-producing capability of bacterial isolates.⁴³ A culture of bacteria was grown in 1% glucose-enriched tryptone soy broth (TSB) (HiMedia) and then transferred to a 96-well polystyrene tissue culture plate (TCP) (Tarsons, India) after being diluted 1:100 with fresh media. Strains of Staphylococcus aureus (ATCC 25923) and Pseudomonas aeruginosa (ATCC 27853) were utilised as positive and sterile TSB as a negative control. The biofilm underwent fixation (with 2% sodium acetate), staining (with 0.1% crystal violet for 5 min), and measurement utilising a micro-ELISA auto reader (Thermo Scientific, Multiskan Go). The experiment was performed three times.

The interpretation of the results was carried out in accordance with Stepanović et al⁴⁴ Average optical density (OD) values were calculated for all tested bacterial isolates and the negative control. We referred to the average OD of the bacterial isolates as (ODei), while that of the negative control was (ODnc). The results from the test were interpreted as follows: if the average ODei value was less than or equal to ODnc, it indicated that the bacteria did not produce biofilm. Conversely, if the average ODei value exceeded ODnc, it indicated that the bacteria produced biofilm.⁴⁵

Statistical Analysis

All data were analysed using Jamovi software version 2.4.8.0. The normality of the data was evaluated with the Shapiro-Wilk test. Measurement results were non-normal and provided as median (IQR). Vitamin D status in DFI patients was compared to clinical, biochemical, inflammatory, and microbiological characteristics using the Kruskal-Wallis test. Dwass-Steel-Critchlow-Fligner (DSCF) was employed for post-hoc analysis. The chi-square test analysed relationships among categorical variables. Spearman's correlation test examined DFI patients’ vitamin D levels and other parameters.

Results

Baseline Characteristics of Patients with DFI

The median age of the 169 patients with DFI was 59 years, and the majority of them were male (86.9%). Most participants (79.9%) adhered to a mixed diet, and the median body mass index was 22 kg/m², with an IQR of 18 to 26.1. Approximately 48.5% reported consuming alcohol, whereas 40.2% identified as smokers. The median diabetes duration in this cohort was 10 years. 74.6% had a prior episode of DFI, while 25.4% had a history of amputation. Among the DFI patients, merely 28.4% exhibited comprehension of appropriate foot care and hygiene protocols. The median duration of ulcers was 23 days, with patients generally encountering between 1 and 2 ulcers. The median dimensions of the ulcers were 2 cm in both length and width, and the median duration of hospitalisation for diabetic foot infection treatment was 8 days. The HbA1c level was 9%, signifying inadequate regulation of blood glucose levels. The median concentrations of VDR protein and LL-37 were 0.832 ng/mL and 1.54 ng/mL, respectively. The median serum vitamin D concentration was 15.3 ng/mL, with 70.4% of DFI patients categorised as vitamin D deficient, 19.5% as insufficient, and 10.1% as sufficient. The median WBC count was 11.8 × 10³/μL. The median counts for neutrophils, lymphocytes, and platelets were 7.3 × 10³/μL, 1.37 × 10³/μL, and 405 × 10³/μL, respectively. The median NLR was 5.27, and the median SII was 2323, signifying overall inflammation in these patients. Table 1 outlines the baseline characteristics of patients with DFI.

Table 1.

Baseline Characteristics of Patients with Diabetic Foot Infections (N = 169).

Parameters	Median (IQR)/Number (%)
Age (years)	59 (54-68)
Gender
Male	147 (86.9%)
Female	22 (13.0%)
Type of food intake
Vegetarian	34 (20.1%)
Mixed	135 (79.9%)
BMI (Kg/m²)	22 (18-26.1)
Alcohol abuse
Yes	82 (48.5%)
No	87 (51.5%)
Smoking
Yes	68 (40.2%)
No	101 (59.8%)
Duration of T2DM (years)	10 (7-14)
Diabetic foot history
Previous ulceration	126 (74.6%)
Previous amputation	43 (25.4%)
Information on foot care in DFI patients
Yes	48 (28.4%)
No	121 (71.6%)
Duration of the existing ulcer (days)	23 (19-27)
Number of foot ulcers, currently	2 (1-2)
Size of current ulcers
Length (cm)	2 (2-3)
Breadth (cm)	2 (2-3)
Length of hospitalisation due to DFI (days)	8 (7-9)
HbA1c (%)	9 (7.8-10.5)
VDR protein (ng/mL)	0.832 (0.575-1.12)
LL-37(ng/mL)	1.54 (1.07-2.13)
Vitamin D status
Deficient (<20 ng/mL)	119 (70.4%)
Insufficient (21-29 ng/mL)	33 (19.5%)
Sufficient (>30 ng/mL)	17 (10.1%)
WBC (×10³/µL)	11.8 (9-13.9)
CRP (mg/L)	64.5 (23-119)
Neutrophils (×10³/µL)	7.3 (5.1-9.7)
Lymphocytes (×10³/µL)	1.37 (1.13-1.63)
Platelets (×10³/µL)	405 (277-652)
SII	2323 (869-3886)
NLR	5.27 (2.75-6.89)

BMI, Body mass index; CRP, C-reactive protein; DFI, Diabetic foot infection; HbA1c, Glycated haemoglobin; IQR, Interquartile range; NLR, Neutrophil-lymphocyte ratio; N, Number of patients; SII, Systemic immune-inflammation index; T2DM, Type 2 diabetes mellitus; WBC, White blood cells; VDR, Vitamin D receptor; LL-37, Cathelicidin.

Association of Serum Vitamin D Status with Clinical, Biochemical, Inflammatory, and Microbiological Parameters in DFI Patients

Compared to both sufficient and insufficient vitamin D groups, a significant increase in various parameters, such as number of ulcers, ulcer size (length and breadth), duration of existing ulcers, hospital stays, HbA1c levels, WBC count, CRP levels, neutrophils, lymphocytes, NLR, and SII, were observed in the vitamin D-deficient group (P < .001). On the other hand, a significant decrease in the levels of VDR and LL-37 was noted in both the vitamin D-deficient and insufficient groups compared to the sufficient group (P < .001), as shown in Table 2.

Table 2.

Association of Serum Vitamin D Status with Various Clinical, Biochemical, and Inflammatory Parameters in DFI Patients.

Parameters	Deficient (N = 119)	Insufficient (N = 33)	Sufficient (N = 17)	P-Value^b
Clinical parameters^a
Age (years)	59^c	59^c	62^c	.450
Duration of T2DM (years)	10^c	5^c	7^c	.071
Number of ulcers	2^c	2^d	1^d	<.001
Ulcer length (cm)	3^c	2^d	1^d	<.001
Ulcer width (cm)	2^c	1.5^d	1^d	<.001
Duration of current ulcer (days)	25^c	19^d	16^d	<.001
Hospital stays due to DFI (days)	8^c	6^d	5^e	<.001
Biochemical parameters^a
HbA1c (%)	9.3^c	8.1^d	7.8^d	<.001
VDR protein (ng/mL)	0.67^c	1.29^d	1.66^e	<.001
LL-37 (ng/mL)	1.27^c	2.31^d	3.12^e	<.001
Inflammatory parameters^a
WBC (×10³/µL)	12.7^c	9.8^d	8.2^d	<.001
CRP (mg/L)	79^c	19.7^d	25.3^cd	<.001
Neutrophils (×10³/µL)	8.04^c	4.86^d	2.1^d	<.001
Lymphocytes (×10³/µL)	1.4^c	1.21^c	1.02^d	.002
NLR	5.99^c	2.93^d	2.08^d	<.001
SII	2835^c	857^d	462^d	<.001

Data are expressed as median, ^b Kruskal Wallis test followed by the Dwass-Steel-Critchlow-Fligner (DSCF)'s test for the post hoc analysis (in case of three or more groups and data of non-normal distribution), c, d, e: Different superscripts in rows indicate significant differences (P < .05) between groups—If the superscript c, d or e is the same, there is no statistical difference between the groups, CRP, C-reactive protein; DFI, Diabetic foot infection; HbA1c, Glycated haemoglobin; NLR, Neutrophil-lymphocyte ratio; LL-37, Cathelicidin; N, number of patients; SII, Systemic immune-inflammation Index; T2DM, Type 2 diabetes mellitus; VDR, Vitamin D receptor; WBC, White blood cells.

Association of Serum Vitamin D Status with Biofilm-Producing Bacterial Isolates in DFI Patients

The bacteria frequently detected in wound specimens from patients with DFI are methicillin-susceptible Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Klebsiella pneumoniae. Figure 2 shows the percentage of bacterial isolates obtained from these wound samples. A significant majority of bacteria (84.36%) isolated from the wounds of DFI patients with vitamin D deficiency exhibited biofilm production (P < .001), as illustrated in Table 3.

Figure 2.

Bacterial Isolates from Wound Samples of DFI Patients.

Table 3.

Association of Serum Vitamin D Status with Biofilm-Producing Bacterial Isolates in DFI Patients.

Type of Bacteria	Vitamin D Status			P-Value^a
Type of Bacteria	Deficient	Insufficient	Sufficient	P-Value^a
Biofilm-producing bacteria	124 (84.36%)	27 (75%)	05 (29.41%)	<.001
Non-biofilm-producing bacteria	23 (15.64%)	9 (25%)	12 (70.59%)	<.001

Chi-square test.

Correlation of Vitamin D Concentrations with Patient-Related Factors in DFI Patients

Vitamin D levels exhibited a significant negative correlation with ulcer duration, hospital stay due to DFI, WBC count, NLR, CRP, and SII (r = −0.787, −0.849, −0.6, −0.475, −0.402, −0.521, respectively; P < .001), while demonstrating a significant positive correlation with VDR and LL-37 levels (r = 0.988 and 0.944, respectively; P < .001). Additionally, HbA1c levels and lymphocyte counts exhibited a significant weak negative connection with vitamin D concentrations (r = −0.327; P < .001 and r = −0.18; P = .027, respectively). The age did not exhibit a significant correlation (r = 0.011; P = .883) with vitamin D concentration, as shown in Table 4.

Table 4.

Correlation of Vitamin D Concentrations with Patient-Related Factors in DFI Patients.

Variables	Spearman Coefficient (r)	P-Value^a
Age	0.011	.883
Duration of T2DM	−0.223	.004
Duration of existing ulcers	−0.787	<.001
No. of ulcers	−0.449	<.001
Ulcer length	−0.542	<.001
Ulcer width	−0.477	<.001
Hospital stays due to DFI	−0.849	<.001
HbA1c	−0.327	<.001
VDR protein	0.988	<.001
LL-37	0.944	<.001
WBC	−0.6	<.001
Neutrophils	−0.471	<.001
Lymphocytes	−0.18	.027
Platelets	−0.442	<.001
NLR	−0.475	<.001
CRP	−0.402	<.001
SII	−0.521	<.001

Spearman's correlation test, CRP, C-reactive protein; HbA1c, Glycated haemoglobin; NLR, Neutrophil-lymphocyte ratio; LL-37, Cathelicidin; N, Number of patients; SII, Systemic immune-inflammation Index; T2DM, Type 2 diabetes mellitus; VDR, Vitamin D receptor; WBC, White blood cells.

Discussion

The study's primary outcome revealed that, among 169 DFI patients tested for vitamin D levels, a significant majority were vitamin D-deficient (70.4%). This confirms past studies on vitamin D deficiency as a significant risk factor for developing DFI.^3,29,46–48 Nonetheless, certain studies have indicated insufficient -linkage between vitamin D and DFI, which could be due to variations in the methodologies employed for assessing vitamin D levels.^7,49,50 In our study, we employed an electrochemiluminescence immunoassay and assessed vitamin D levels, while the other investigations assessed through the ELISA technique.

A study conducted in China reported that patients with unhealed ulcers were vitamin D-deficient compared to those with healed ulcers, though levels were not statistically significant.⁵¹ Similarly, the current study findings revealed that vitamin D-deficient patients with DFI had significantly bigger ulcer sizes (both in length and breadth), longer durations of ulcers, and extended hospital stays. However, a study conducted in Brazil demonstrated that patients with vitamin D-sufficiency exhibited similar ulcer characteristics to those with vitamin D deficiency.⁵² The researchers suggested that deficiency of this vitamin might not be the primary cause of ulcers but rather a consequence of other factors. Nevertheless, this deficiency could still hinder the healing process.⁵² Their trial results did not reveal a significant difference in ulcer size between the vitamin D groups and the placebo group after treatment; however, there was a trend suggesting potential statistical significance in wound size changes following vitamin D therapy.⁵²

In the present study, most inflammatory parameters, including WBC, CRP, NLR, and SII, were significantly elevated in the vitamin D-deficient group compared to the other vitamin D groups. Notably, the most significant differences in these parameters were observed primarily between the vitamin D-deficient group and the vitamin D-insufficient group. This supports earlier preclinical and clinical research showing vitamin D's anti-inflammatory benefits on inflammatory indicators.^53–55

A northern Chinese study found that vitamin D deficiency indirectly reduced VDR transcription.⁵⁶ Additionally, Mandal et al found that low levels of vitamin D3 are associated with extremely low expression of VDR in individuals exhibiting complicated symptoms of leprosy.²³ These findings are consistent with our results, as we observed significant reductions in the concentrations of VDR and LL-37 in the vitamin D-deficient group, followed by the insufficient group. While the highest levels were found in the vitamin D-sufficient group. A study in tuberculosis patients found no definitive correlation between levels of vitamin D and LL-37.⁵⁷ However, in septic and non-septic ICU patients, a correlation between vitamin D status and levels of LL-37 was established.⁵⁸ The conflicting results indicate that differences in underlying illness conditions may influence the possible relationship between serum vitamin D and LL-37 levels. Another key finding was that HbA1c levels were significantly higher in the vitamin D-deficient group compared to the sufficient group. A similar trend has also been noted in studies conducted in the United States as well as in China.^59,60 Vitamin D's role in glycaemic control and its indirect effects on wound healing may explain this difference. Vitamin D plays a crucial role in enhancing the body's ability to use insulin efficiently and regulate blood sugar levels.⁶¹ This can assist in the management of diabetes and its related problems, like DFIs, by strengthening the immune system, decreasing inflammation, and facilitating tissue repair.^62–64

Our study reveals that a significant majority of bacteria (84.36%) isolated from the wounds of DFI patients with vitamin D deficiency exhibited biofilm production. Individuals with diabetes frequently experience a persistent hyperglycaemic condition, fostering an optimal environment for microbial proliferation.⁶⁵ The bacteria frequently found in wound samples from DFI patients included methicillin-susceptible Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Klebsiella pneumoniae. These microorganisms thrive and form biofilms.^7,25 Biofilms typically consist of extracellular polymeric substances that are generated by stationary microorganisms.⁶⁶ Increased glucose levels promote the production of extracellular polymeric substances, leading microorganisms to shift to a sessile state and integrate into the biofilm matrix, which obstructs antibiotic penetration and diminishes treatment efficacy.⁶⁷ While a recent study indicates that vitamins D and K1 can effectively combat biofilms produced by specific Gram-negative bacteria, suggesting their potential efficacy in improving therapies for infections brought on by strains that are resistant to multiple drugs and biofilm-forming bacteria.⁶⁸ Nonetheless, additional in vivo research is required to substantiate these promising findings.

This study has certain limitations as it was carried out at a single centre with a limited sample size, hindering the ability to establish causality. Additionally, the TCP method employed for the analysis of bacterial biofilms was performed in vitro, which leaves the implications of biofilm presence in vivo still ambiguous. Moreover, numerous limitations inherent in vitamin D research complicate the establishment of definitive conclusions regarding the association between vitamin D status and the risk of diabetic complications. These are considerable differences seen among the studies, including variabilities in patients’ demographics and genetic, baseline vitamin D status, doses of vitamin D supplements, and primary endpoint selection.⁶⁹ It is important to note that the Endocrine Society no longer endorses specific serum vitamin D levels to define sufficiency, insufficiency, and deficiency.⁷⁰

Current clinical guidelines provide only limited support for vitamin D supplementation in the prevention of diabetes among individuals with prediabetes.⁷⁰ Collectively, the study findings showed that the majority of DFI patients exhibited vitamin D deficiency. This significantly impacted most of the clinical, biochemical, and inflammatory parameters, in addition to the microbial environment within the wound, leading to less favourable outcomes in DFI patients. The research highlights the importance of regularly checking vitamin D levels in patients with DFI and providing vitamin D supplements to address deficiencies. This approach aims to prevent recurrent infections and reduce the risk of amputations. Considering the clinical implications of this study, subsequent investigations should focus on implementing real-time testing to evaluate how the restoration of vitamin D levels affects the patient-related parameters in DFI patients. Therefore, the study suggests implementing a rigorously controlled, extensive, multicentric randomised trial utilising vitamin D supplements to assess the impact of restoring vitamin D levels on the outcome measures in patients with DFI.

Footnotes

Acknowledgments

The authors would like to express their gratitude to the Department of Health Research, Ministry of Health & Family Welfare, New Delhi, India, for providing funding through the grant-in-aid scheme (No.R.11014/31/2023-GIA/HR). Additionally, we thank the Manipal Academy of Higher Education, Manipal, India, for offering research support. We also appreciate the Department of Microbiology at Kasturba Medical College, Manipal, for providing the laboratory facilities necessary for sample processing. Dr. Saleena Ummer Velladath contributed to the research while affiliated with Manipal Academy of Higher Education, Manipal. Dr. Velladath‘s current affiliation mentioned is for correspondence purposes only. No foreign collaboration, funding. or material transfer were involved in this work.

ORCID iDs

Ruby Benson

Shilia Jacob Kurian

Seetharam Shiva Prasad

Barnini Banerjee

Varun Kumar Sudha Gururaj

Vijayanarayana Kunhikatta

Saleena Ummer Velladath

Gabriel Sunil Rodrigues

Raghu Chandrashekar Hariharapura

Mahadev Rao

Murali Munisamy

Shashidhar Vishwanath

Chiranjay Mukhopadhyay

Sonal Sekhar Miraj

Ethics Approval & Trial Registry Details

The study received approval from the Institutional Ethics Committee of Kasturba Medical College & Kasturba Hospital, Manipal, India, with reference no. IEC 676/2020 and was executed in accordance with the Helsinki Declaration 2013. Written informed consent was obtained from the patients after providing patient information sheets and ensuring the patients understood the objectives and purpose of the study. The study has been registered in the Clinical Trial Registry of India (CTRI) with registration number CTRI/2021/02/031281. Animal studies: N/A.

Authors' Contributions

S.S.M. and R.B. were involved in the conceptualization and design of the study. R.B. was involved in data collection for this study. R.B., S.J.K., S.S.P., B.B., and S.V. were involved in case diagnosis and data interpretation for this article. R.B., B.B., and S.V. were involved in processing the samples. V.K.S.G. and V.K. were involved in data analysis. R.B. and S.S.M. were involved in manuscript writing. S.S.P., S.U.V., G.S.R., R.C.H., M.R., C.M., and M.M. were involved in critically reviewing this article. S.S.M., S.U.V., M.R., B.B, C.M., and R.C.H. were involved in the acquisition of the funding for the study. All the authors approved the final draft of the manuscript.

Declaration of Conflicting Interests

The authors declare no conflict of interest. The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony or patents received or pending, or royalties. The funding sources were not involved in the study design, the collection, analysis, and interpretation of data in the writing of the report, or in the decision to submit the article for publication.

Data Availability Statement

Upon reasonable request, the corresponding author will provide the supporting data for the study.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the Grant-in-aid (GIA) grant (No. R.11014/31/2023-GIA/HR), Department of Health Research, Ministry of Health & Family Welfare, New Delhi, India.

References

International Diabetes Federation . IDF Diabetes Atlas. 11th ed. International Diabetes Federation; 2025 [cited 2025 May 28]. https://diabetesatlas.org

Anjana

Unnikrishnan

Deepa

, et al. Metabolic non-communicable disease health report of India: the ICMR-INDIAB national cross-sectional study (ICMR-INDIAB-17). Lancet Diabetes Endocrinol. 2023;11(7):474–489. doi:10.1016/S2213-8587(23)00119-5

Priyanto

Legiawati

Saldi

SRF

Yunir

Miranda

. Comparison of vitamin D levels in diabetes mellitus patients with and without diabetic foot ulcers: an analytical observational study in Jakarta, Indonesia. Int Wound J. 2023;20(6):2028–2036. doi:10.1111/iwj.14066

Edmonds

Manu

Vas

. The current burden of diabetic foot disease. J Clin Orthop Trauma. 2021;17:88–93. doi: 10.1016/j.jcot.2021.01.017

Akkus

Sert

. Diabetic foot ulcers: a devastating complication of diabetes mellitus continues non-stop in spite of new medical treatment modalities. World J Diabetes. 2022;13(12):1106–1121. doi:10.4239/wjd.v13.i12.1106

Raghav

Khan

Labala

Ahmad

Noor

Mishra

. Financial burden of diabetic foot ulcers to world: a progressive topic to discuss always. Ther Adv Endocrinol Metab. 2018;9(1):29–31. doi:10.1177/2042018817744513

Kurian

Benson

Munisamy

, et al. Plasma vitamin D status and its association with biochemical, clinical and humanistic outcomes in diabetic foot infection patients: a prospective observational study in a tertiary healthcare facility. Expert Rev Endocrinol Metab. 2025;20(3):233–239. doi:10.1080/17446651.2025.2480374

Wang

Meng

, et al. The awareness and determinants of diabetic foot ulcer prevention among diabetic patients: insights from NHANES (2011-2018). Prev Med Rep. 2023;36:102433. doi:10.1016/j.pmedr.2023.102433

Wen

Dong

, et al. The metabolic characteristics of patients at the risk for diabetic foot ulcer: a comparative study of diabetic patients with and without diabetic foot. Diabetes Metab Syndr Obes. 2023;16:3197–3211. doi:10.2147/DMSO.S430426

10.

Akyüz

Bahçecioğlu Mutlu

Guven

Başak

Yilmaz

. Elevated HbA1c level associated with disease severity and surgical extension in diabetic foot patients. Diyabetik ayak hastalarında yüksek HbA1c düzeyi ile hastalık şiddeti ve cerrahi seviye ilişkisi. Ulus Travma Acil Cerrahi Derg. 2023;29(9):1013–1018. doi:10.14744/tjtes.2023.08939

11.

Zhang

Tang

, et al. C-reactive protein and diabetic foot ulcer infections: a meta-analysis. J Tissue Viability. 2022;31(3):537–543. doi:10.1016/j.jtv.2022.05.001

12.

Sathvik

Vuppuluri

Dulipala

. The association of the neutrophil-lymphocyte ratio with the outcome of diabetic foot ulcer. Cureus. 2023;15(1):e33891. doi:10.7759/cureus.33891

13.

Aydın

Eren

Uyar

, et al. Relationship between systemic immune inflammation index and amputation in patients with diabetic foot ulcer. J Orthop Sci. 2024;29(4):1060–1063. doi:10.1016/j.jos.2023.07.015

14.

Afonso

Oliveira

Saavedra

Borges

Simões

. Biofilms in diabetic foot ulcers: impact, risk factors and control strategies. Int J Mol Sci. 2021;22(15):8278. Published 2021 Jul 31. doi:10.3390/ijms22158278

15.

Voltan

Cannito

Ferrarese

Ceccato

Camozzi

. Vitamin D: an overview of gene regulation, ranging from metabolism to genomic effects. Genes (Basel). 2023;14(9):1691. doi:10.3390/genes14091691

16.

Janoušek

Pilařová

Macáková

, et al. Vitamin D: Sources, physiological role, biokinetics, deficiency, therapeutic use, toxicity, and overview of analytical methods for detection of vitamin D and its metabolites. Crit Rev Clin Lab Sci. 2022;59(8):517–554. doi:10.1080/10408363.2022.2070595

17.

Lei

Liu

Guo

. The emerging role of vitamin D and vitamin D receptor in diabetic nephropathy. Biomed Res Int. 2020;2020:4137268. doi:10.1155/2020/4137268

18.

Bishop

Ismailova

Dimeloe

Hewison

White

. Vitamin D and immune regulation: antibacterial, antiviral, anti-inflammatory. JBMR Plus. 2020;5(1):e10405. doi:10.1002/jbm4.10405

19.

Charoenngam

Holick

. Immunologic effects of vitamin D on human health and disease. Nutrients. 2020;12(7):2097. doi:10.3390/nu12072097

20.

Yang

Good

Mosaiab

, et al. Significance of LL-37 on immunomodulation and disease outcome. Biomed Res Int. 2020;2020:8349712. doi:10.1155/2020/8349712

21.

Medeiros

JFP

de Oliveira Borges

Soares

, et al. The impact of vitamin D supplementation on VDR gene expression and body composition in monozygotic twins: randomized controlled trial. Sci Rep. 2020;10(1):11943. doi:10.1038/s41598-020-69128-2

22.

Gombart

Borregaard

Koeffler

. Human cathelicidin antimicrobial peptide (CAMP) gene is a direct target of the vitamin D receptor and is strongly up-regulated in myeloid cells by 1,25-dihydroxyvitamin D3. FASEB J. 2005;19(9):1067–1077. doi: 10.1096/fj.04-3284com

23.

Mandal

Reja

Biswas

Bhattacharyya

Patra

Bhattacharya

. Vitamin D receptor expression levels determine the severity and complexity of disease progression among leprosy reaction patients. New Microbes New Infect. 2015;6:35–39. doi:10.1016/j.nmni.2015.04.001

24.

Zhao

Pan

, et al. Expression and significance of serum vitamin D and LL-37 levels in infants with bacterial pneumonia. Front Pediatr. 2022;10:989526. doi:10.3389/fped.2022.989526

25.

Benson

Unnikrishnan

Kurian

, et al. Vitamin D attenuates biofilm-associated infections via immunomodulation and cathelicidin expression: a narrative review. Expert Rev Anti Infect Ther. 2023;21(1):15–27. doi:10.1080/14787210.2023.2151439

26.

Lungu

Kilembe

Lakhi

, et al. A comparison of vitamin D and cathelicidin (LL-37) levels between patients with active TB and their healthy contacts in a high HIV prevalence setting: a prospective descriptive study. Trans R Soc Trop Med Hyg. 2022;116(4):336–343. doi:10.1093/trstmh/trab126

27.

Pouget

Dunyach-Remy

Pantel

Schuldiner

Sotto

Lavigne

. Biofilms in diabetic foot ulcers: significance and clinical relevance. Microorganisms. 2020;8(10):1580. doi:10.3390/microorganisms8101580

28.

Gombart

. The vitamin D-antimicrobial peptide pathway and its role in protection against infection. Future Microbiol. 2009;4(9):1151–1165. doi:10.2217/fmb.09.87

29.

Dai

Jiang

Chen

Chai

. Vitamin D and diabetic foot ulcer: a systematic review and meta-analysis. Nutr Diabetes. 2019;9(1):8. doi:10.1038/s41387-019-0078-9

30.

Iqhrammullah

Duta

Alina

, et al. Role of lowered level of serum vitamin D on diabetic foot ulcer and its possible pathomechanism: a systematic review, meta-analysis, and meta-regression. Diabetes Epidemiol Manag. 2024;13:100175. doi:10.1016/j.deman.2023.100175

31.

Zhou

Hyppönen

. Vitamin D deficiency and C-reactive protein: a bidirectional Mendelian randomization study. Int J Epidemiol. 2023;52(1):260–271. doi:10.1093/ije/dyac087

32.

Wang

Shen

Zhang

. Vitamin D affects the neutrophil-to-lymphocyte ratio in patients with type 2 diabetes mellitus. J Diabetes Investig. 2021;12(2):254–265. doi:10.1111/jdi.13338

33.

Chan

Cui

Peng

Liu

. The associations among serum vitamin D concentration, systemic immune-inflammation index, and lifestyle factors in Chinese adults: a cross-sectional analysis. Front Nutr. 2025;12:1543925. doi:10.3389/fnut.2025.1543925

34.

Ghaferi

Schwartz

Pawlik

. STROBE Reporting guidelines for observational studies. JAMA Surg. 2021;156(6):577–578. doi:10.1001/jamasurg.2021.0528

35.

Senneville

Albalawi

van Asten

, et al. IWGDF/IDSA guidelines on the diagnosis and treatment of diabetes-related foot infections (IWGDF/IDSA 2023). Clin Infect Dis. 2023. doi:10.1093/cid/ciad527

36.

Dai

Chen

Chai

. Association between serum 25-OH-vitamin D and diabetic foot ulcer in patients with type 2 diabetes. Front Nutr. 2020;7:109. doi:10.3389/fnut.2020.00109

37.

Tang

Chen

, et al. Association between vitamin D status and diabetic foot in patients with type 2 diabetes mellitus. J Diabetes Investig. 2022;13(7):1213–1221. doi:10.1111/jdi.13776

38.

Lipsky

Tabak

Johannes

Hyde

Weigelt

. Skin and soft tissue infections in hospitalised patients with diabetes: Culture isolates and risk factors associated with mortality, length of stay and cost. Diabetologia. 2010;53(5):914–923. doi:10.1007/s00125-010-1672-5

39.

Holick

Binkley

Bischoff-Ferrari

, et al. Evaluation, treatment, and prevention of vitamin D deficiency: an Endocrine Society clinical practice guideline. J Clin Endocrinol Metab. 2011;96(7):1911–1930. doi:10.1210/jc.2011-0385

40.

Iriani

Rachman

Fatina

, et al. Vitamin D status, vitamin D receptor, CYP2R1, and CYP24A1 profiles in children. Front Nutr. 2024;11:1394367. doi:10.3389/fnut.2024.1394367

41.

Sui

. Polyaminoglycoside nanosystem expressing antimicrobial peptides for multistage chronic wound management. J Control Release. 2025;382:113657. doi:10.1016/j.jconrel.2025.113657

42.

Harika

Shenoy

Narasimhaswamy

Chawla

. Detection of biofilm production and its impact on antibiotic resistance profile of bacterial isolates from chronic wound infections. J Glob Infect Dis. 2020;12(3):129–134. doi:10.4103/jgid.jgid_150_19

43.

Christensen

Simpson

Younger

, et al. Adherence of coagulase-negative staphylococci to plastic tissue culture plates: a quantitative model for the adherence of staphylococci to medical devices. J Clin Microbiol. 1985;22(6):996–1006. doi:10.1128/jcm.22.6.996-1006.1985

44.

Stepanović

Vuković

Hola

, et al. Quantification of biofilm in microtiter plates: overview of testing conditions and practical recommendations for assessment of biofilm production by staphylococci. APMIS. 2007;115(8):891–899. doi:10.1111/j.1600-0463.2007.apm_630.x

45.

Kabir

Ahsan

Rahman

Jobayer

Shamsuzzaman

. Biofilm-producing and specific antibiotic resistance genes in Pseudomonas aeruginosa isolated from patients admitted to a tertiary care hospital, Bangladesh. IJID Reg. 2024;11:100369. doi: 10.1016/j.ijregi.2024.100369

46.

Tiwari

Pratyush

Gupta

, et al. Prevalence and severity of vitamin D deficiency in patients with diabetic foot infection. Br J Nutr. 2013;109(1):99–102. doi:10.1017/S0007114512000578

47.

Tang

Chen

, et al. The correlation between serum vitamin D status and the occurrence of diabetic foot ulcers: a comprehensive systematic review and meta-analysis. Sci Rep. 2024;14(1):21932. doi:10.1038/s41598-024-73133-0

48.

Tang

Huang

Luo

, et al. Level of 25-hydroxyvitamin D and vitamin D receptor in diabetic foot ulcer and factor associated with diabetic foot ulcers. Diabetol Metab Syndr. 2023;15(1):30. doi:10.1186/s13098-023-01002-3

49.

Hammad

Abdel Wahab

Farouk

, et al. Non-classical monocytes frequency and serum vitamin D3 levels are linked to diabetic foot ulcer associated with peripheral artery disease. J Diabetes Investig. 2023;14(10):1192–1201. doi:10.1111/jdi.14048

50.

Afarideh

Ghanbari

Noshad

Ghajar

Nakhjavani

Esteghamati

. Raised serum 25-hydroxyvitamin D levels in patients with active diabetic foot ulcers. Br J Nutr. 2016;115(11):1938–1946. doi:10.1017/S0007114516001094

51.

Tang

Chen

, et al. Association of vitamin D status with all-cause mortality and outcomes among Chinese individuals with diabetic foot ulcers. J Diabetes Investig. 2023;14(1):122–131. doi:10.1111/jdi.13917

52.

Burkiewicz

Guadagnin

Skare

do Nascimento

Servin

de Souza

. Vitamin D and skin repair: a prospective, double-blind and placebo controlled study in the healing of leg ulcers. Rev Col Bras Cir. 2012;39(5):401–407. doi:10.1590/s0100-69912012000500011

53.

Agh

Mousavi

Aryaeian

, et al. Protective effect of vitamin D supplementation on some hemogram derived inflammatory indices in normal and high-fat diet fed male wistar rats. Int J Prev Med. 2023;14:49. doi:10.4103/ijpvm.ijpvm_505_20

54.

de Oliveira

Biddulph

Hirani

Schneider

IJC

. Vitamin D and inflammatory markers: cross-sectional analyses using data from the English Longitudinal Study of Ageing (ELSA). J Nutr Sci. 2017;6:e1. doi:10.1017/jns.2016.37

55.

Zhao

Fang

Zhou

Zhang

Peng

. The relationship between novel inflammatory markers and serum 25-hydroxyvitamin D among US adults. Immun Inflamm Dis. 2025;13(1):e70115. doi:10.1002/iid3.70115

56.

Lin

, et al. Increase of circulating cholesterol in vitamin D deficiency is linked to reduced vitamin D receptor activity via the Insig-2/SREBP-2 pathway. Mol Nutr Food Res. 2016;60(4):798–809. doi:10.1002/mnfr.201500425

57.

Yamshchikov

Kurbatova

Kumari

, et al. Vitamin D status and antimicrobial peptide cathelicidin (LL-37) concentrations in patients with active pulmonary tuberculosis. Am J Clin Nutr. 2010;92(3):603–611. doi:10.3945/ajcn.2010.29411

58.

Jeng

Yamshchikov

Judd

, et al. Alterations in vitamin D status and anti-microbial peptide levels in patients in the intensive care unit with sepsis. J Transl Med. 2009;7:28. doi:10.1186/1479-5876-7-28

59.

Ford

Zhao

Tsai

. Associations between concentrations of vitamin D and concentrations of insulin, glucose, and HbA1c among adolescents in the United States. Diabetes Care. 2011;34(3):646–648. doi:10.2337/dc10-1754

60.

Zhao

Zhen

Wang

, et al. The relationship between vitamin D deficiency and glycated hemoglobin levels in patients with type 2 diabetes mellitus. Diabetes Metab Syndr Obes. 2020;13:3899–3907. doi:10.2147/DMSO.S275673

61.

Razzaghi

Pourbagheri

Momen-Heravi

, et al. The effects of vitamin D supplementation on wound healing and metabolic status in patients with diabetic foot ulcer: a randomized, double-blind, placebo-controlled trial. J Diabetes Complications. 2017;31(4):766–772. doi:10.1016/j.jdiacomp.2016.06.017

62.

Gong

, et al. Effects of vitamin D status on cutaneous wound healing through modulation of EMT and ECM. J Nutr Biochem. 2024;134:109733. doi:10.1016/j.jnutbio.2024.109733

63.

Tang

Chen

Zhang

Ran

. The multi-dimensional role of vitamin D in the pathophysiology and treatment of diabetic foot ulcers: From molecular mechanisms to clinical translation. Int J Mol Sci. 2025;26(12):5719. doi:10.3390/ijms26125719

64.

Greenhagen

Frykberg

Wukich

. Serum vitamin D and diabetic foot complications. Diabet Foot Ankle. 2019;10(1):1579631. doi:10.1080/2000625X.2019.1579631

65.

Darwitz

Genito

Thurlow

. Triple threat: How diabetes results in worsened bacterial infections. Infect Immun. 2024;92(9):e0050923. doi:10.1128/iai.00509-23

66.

Ray

Weis

Nwaeze

Zhou

Basu

Mitra

. Development and control of biofilms in diabetic foot infections: a narrative review. Acta Microbiologica Hellenica. 2025;70(1):9. doi:10.3390/amh70010009

67.

Sahoo

Meshram

. Biofilm formation in chronic infections: a comprehensive review of pathogenesis, clinical implications, and novel therapeutic approaches. Cureus. 2024;16(10):e70629. doi:10.7759/cureus.70629

68.

Lutfi

Shaaban

Elshaer

. Vitamin D and vitamin K1 as novel inhibitors of biofilm in gram-negative bacteria. BMC Microbiol. 2024;24(1):173. doi:10.1186/s12866-024-03293-6

69.

Vasdeki

Tsamos

Dimakakos

, et al. Vitamin D supplementation: shedding light on the role of the sunshine vitamin in the prevention and management of type 2 diabetes and its complications. Nutrients. 2024;16(21):3651. doi: 10.3390/nu16213651

70.

Demay

Pittas

Bikle

, et al. Vitamin D for the prevention of disease: an endocrine society clinical practice guideline. J Clin Endocrinol Metab. 2024;109(8):1907–1947. doi:10.1210/clinem/dgae290

Association of Serum Vitamin D Status with Multidimensional Health Parameters in Patients with Diabetic Foot Infections: A Cross-Sectional Analysis in a Tertiary Healthcare Facility

Abstract

Aims/Introduction

Materials and Methods

Results

Conclusions

Keywords

Introduction

Methods

Study Design and Ethical Approval

Patient Enrolment

Data Collection Methods

Measurement of Serum Vitamin D Levels in DFI Patients

Estimation of Vitamin D Receptor and LL-37 Levels in Serum Samples of DFI Patients

Analysis of Biofilm Formation in Bacterial Isolates from Wound Samples of DFI Patients

Statistical Analysis

Results

Baseline Characteristics of Patients with DFI

Association of Serum Vitamin D Status with Clinical, Biochemical, Inflammatory, and Microbiological Parameters in DFI Patients

Association of Serum Vitamin D Status with Biofilm-Producing Bacterial Isolates in DFI Patients

Correlation of Vitamin D Concentrations with Patient-Related Factors in DFI Patients

Discussion

Footnotes

Acknowledgments

ORCID iDs

Ethics Approval & Trial Registry Details

Authors' Contributions

Declaration of Conflicting Interests

Data Availability Statement

Funding

References