Asymptomatic bacteriuria (ASB) is a prevalent condition in Type-2 Diabetes Mellitus (T2DM) patients, often exacerbated by the chronic inflammatory milieu associated with diabetes. This study aims to evaluate and compare the levels of key inflammatory biomarkers—Interleukin-6 (IL-6), Tumor Necrosis Factor-alpha (TNF-α), C-reactive protein (CRP), and Hemoglobin A1c (HbA1c)—in T2DM patients with and without ASB in Northern Andhra Pradesh. IL-6 and TNF-α are pivotal pro-inflammatory cytokines that contribute to both the pathogenesis of insulin resistance and the inflammatory response during bacterial infections. Elevated IL-6 and TNF-α levels are associated with poor glycemic control and increased susceptibility to infections, including urinary tract infections. CRP, an acute-phase reactant, is commonly elevated in systemic inflammation and serves as a reliable marker for infection and inflammation, which is critical in assessing the risk of ASB in diabetic patients. HbA1c, reflecting long-term blood glucose control, is also implicated in chronic inflammation, with poorly controlled diabetes being linked to higher inflammation and infection rates. By assessing these biomarkers, this study aims to elucidate the relationship between systemic inflammation, glycemic control, and the incidence of ASB in T2DM.
Type-2 Diabetes Mellitus (T2DM) is a chronic metabolic disorder characterized by insulin resistance and hyperglycemia, which predisposes individuals to various infections, including urinary tract infections (UTIs). One such infection, Asymptomatic Bacteriuria (ASB), is the presence of bacteria in the urine without clinical symptoms. ASB is prevalent among diabetic patients, with studies indicating a higher incidence in this population due to impaired immune function and chronic inflammation associated with T2DM1. ASB can lead to complications such as symptomatic urinary tract infections, pyelonephritis, and renal damage if left untreated. Therefore, early detection and management are crucial, especially in individuals with poorly controlled diabetes2.
Chronic inflammation plays a central role in both the pathogenesis of T2DM and the increased susceptibility to infections. Key inflammatory biomarkers, such as Interleukin-6 (IL-6), Tumor Necrosis Factor-alpha (TNF-α), C-reactive protein (CRP), and Hemoglobin A1c (HbA1c), have been implicated in the dysregulated immune response seen in T2DM. IL-6 and TNF-α are pro-inflammatory cytokines that mediate the inflammatory response and contribute to insulin resistance, which further exacerbates glycemic control3. CRP is an acute-phase reactant that is elevated in response to systemic inflammation, making it a valuable marker for detecting infection and inflammation. HbA1c, a marker of long-term glycemic control, reflects the degree of hyperglycemia over time and is often associated with increased inflammation and higher infection risk4.
Although the link between these biomarkers and infection in T2DM is well-documented, limited studies specifically explore their role in ASB among diabetics5. This study aims to evaluate the levels of IL-6, TNF-α, CRP, and HbA1c in T2DM patients with and without ASB in Northern Andhra Pradesh, providing insights into the inflammatory pathways that may contribute to the higher prevalence of ASB in diabetic individuals.
The study was carried out at Gayatri Vidya Parishad Institute of Health Care and Medical Technology, a teaching hospital in Visakhapatnam, Andhra Pradesh, India. A total of 258 Type-2 Diabetes Mellitus (T2DM) patients, aged between 30 and 60 years, participated in the study with exclusion of immunocompromised individuals, patients with symptomatic urinary tract infections (UTIs) and on antimicrobial therapy, those with obstructive uropathy, renal failure, indwelling catheters, and pregnant women. The study was approved by the Institutional Ethical Committee (GVPIHCMT/IEC/20201208/03), and informed consent was obtained from all participants before sample collection.
Testing for ASB
Urine samples from all participants in the study were collected according to standard protocols. The samples were then plated onto MacConkey agar, nutrient agar, and blood agar, and incubated at 37°C for 18-24 hours. If any growth was observed, standard microbiological techniques were employed to identify the organism.
HbA1c estimation
HbA1c was quantified in whole blood using Fluorescent Immuno Chromatographic (Finecare F2071720C AD). The estimations were done following the manufacturer’s instructions.
Estimation of C-Reactive protein (CRP)
CRP was quantified in serum using Fluorescent Immuno Chromatographic (Finecare F2011840D) method. The estimations were done following the manufacturer’s instructions.
Estimation of Cytokines
The serum obtained from patients were stored in -80⁰C and were used for estimation of IL-6; Interlukin-6 (BioLegend, USA, 430501), IL-10; Interlukin-10 (BioLegend, USA, 430601), TNF-α; Tumor Necrosis Factor-α (BioLegend, USA, 430201) and CRP (BioLegend, USA, 430201) as per manufacturer instructions.
Statistical analysis
Samples were analysed in duplicates. Values expressed as mean ± Standard Error Mean (SEM). Data was analysed and graphical representations were plotted using MS Excel. Statistical significance was calculated using t-test.
Testing for ASB
A total of 258 T2DM patients included in the study, 124 were female and 134 were male. Growth was observed in 93 (i.e. 36% of total) specimens of which, females were the majority (61.3%).
HbA1c estimation
A total of 168 subjects (65.1%) had poor glycaemic control with HBA1C > 6.5 of which 73 (43.45%)
HbA1c |
Sterile |
ASB |
Total |
||
Number |
% |
Number |
% |
||
≤ 6.5 |
70 |
77.08 |
20 |
22.22 |
90 |
> 6.5 |
95 |
56.55 |
73 |
43.45 |
168 |
Total |
165 |
63.95 |
93 |
36.05 |
258 |
Table 1 showing HbA1c levels in ASB positive and non-ASB patients. ASB- Asymptomatic Bacteriuria.
Culture positivity among ASB: Among 258, 93 (36%) of ASB yielded significant growth i.e, 105cfu/ml, during initial study. 44% (41) samples yielded growth of Klebsiella spp followed by 28% (26) E.coli, 23% (21) yielded S.aureus and 5% (5) yielded growth of Emterococcus spp.
In the followup, of 93 subjects included, 24 (26%) of clinical samples were determined to be as ASB yielding significant growth as defined above. Of 93 samples, 12 (50%) of samples yielded Klebsiella spp followed by 8 (33%) of E.coli, , 3(13%) of S.aureus and 1 (4%) of Enterococcus spp.
Estimation of Cytokines
Cytokines levels were measured in the serum of the patients in both non-ASB and ASB groups.
IL-6 estimation
IL-6 levels increased significantly (p<0.005) in patients infected with Klebsiella (129.91± 24.00 pg/ml) and S. aureus (109.40 ± 12.52 pg/ml) when compared to patients not having ASB (36.09 ± 5.74 pg/ml). It is also to be noted that patients infected with Enterococci and E. coli did not show any significant difference in IL-6 levels compared to non-ASB group (Table 2).
IL-10 estimation
IL-10 levels were significantly (p<0.005) increased in patients infected with Enterococci (9.17± 0.05 pg/ml) when compared to patients not having ASB (6.90 ± 0.82 pg/ml). Other ASB groups did not show any significant difference in IL-10 levels compared to non-ASB group (Table 2).
TNF-α estimation
TNF-α levels increased significantly (p<0.05) in patients infected with E. coli (59.13 ± 6.26 pg/ml) and S. aureus (51.34 ± 1.04 pg/ml) when compared to patients not having ASB (41.15 ± 5.71 pg/ml). Other ASB positive groups did not show any significant difference in TNF-α levels compared to non-ASB group (Table 2).
CRP estimation
When compared with Non ASB and ASB group, Non ASB group did not show any significant elevation of CRP both in initial and followup period whereas in ASB group a significant elevation of CRP was observed. Among the organisms isolated, infection with Klebsiella spp had a range of 6.83 to 8.44 both in initial and followup, followed by Enterococci with 8.3, E.coli with 6.77 to 7.78 and S.aureus with 5.21 to 6.40 indicating a significant inflammation in ASB (Table 2)
Table 2 showing comparison of biomarkers in ASB and Non ASB groups.
The results of this study emphasize the significant presence of asymptomatic bacteriuria (ASB) in Type 2 Diabetes Mellitus (T2DM) patients and its association with altered inflammatory cytokine profiles. The 36% prevalence of ASB in our cohort is consistent with previous studies, which have reported a higher incidence of ASB in diabetic populations compared to non-diabetic individuals, likely due to the altered immune response and urological changes that are common in diabetes6,7. Our study found that Klebsiella spp. was the most prevalent pathogen, followed by E. coli, S. aureus, and Enterococcus spp. This reflects findings from earlier studies that highlight Klebsiella and E. coli as the dominant pathogens in diabetic patients with ASB8,9. Notably, our follow-up analysis showed that Klebsiella spp. persisted as the most common organism causing ASB, confirming its role as a key pathogen in this condition.
The cytokine analysis revealed significant differences in the levels of pro-inflammatory cytokines such as IL-6 and TNF-α among ASB-positive patients compared to those without ASB. IL-6, a key marker of inflammation, was significantly elevated in patients infected with Klebsiella and S. aureus. This finding corroborates previous research indicating that Klebsiella infection, in particular, induces a potent inflammatory response marked by high levels of IL-6. IL-6 is a major mediator in the acute-phase response, and its overproduction is associated with both tissue damage and the exacerbation of systemic inflammation, which can lead to complications such as nephropathy and cardiovascular disease in diabetic patients10,11. Interestingly, we did not observe significant increases in IL-6 levels in patients infected with E. coli or Enterococcus spp., which may suggest a differential immune response based on the type of pathogen. Some studies have also shown that the inflammatory response to E. coli may be less robust than that triggered by other pathogens, such as Klebsiella, which could explain the absence of a significant IL-6 rise in our study12.
The elevation of TNF-α in patients infected with E. coli and S. aureus further supports the involvement of this cytokine in the inflammatory process during ASB. TNF-α is a critical mediator of immune responses to bacterial infections and has been linked to the pathogenesis of various inflammatory diseases, including diabetic complications13. Our findings are consistent with previous studies that have reported elevated TNF-α levels in bacterial infections, particularly with S. aureus and E. coli 14. The presence of increased TNF-α in the ASB group, along with the elevated IL-6, suggests that these cytokines contribute to the inflammatory milieu in T2DM patients, potentially exacerbating the underlying chronic inflammation associated with diabetes.
IL-10, an anti-inflammatory cytokine, was significantly increased only in patients infected with Enterococcus spp., contrasting with the lack of significant changes in IL-6 and TNF-α in this group. IL-10 is known for its role in regulating immune responses and limiting excessive inflammation15. The elevation of IL-10 in Enterococcus-infected patients may reflect a compensatory response aimed at counterbalancing the pro-inflammatory effects of other cytokines, but its isolated increase in this group warrants further investigation to better understand its role in ASB.
The CRP levels, a marker of systemic inflammation, were significantly elevated in the ASB group compared to the non-ASB group, further supporting the notion that ASB contributes to chronic low-grade inflammation in T2DM patients. Our study demonstrated that infections with Klebsiella were associated with the highest CRP values, a finding consistent with the literature16. The persistent elevation of CRP in ASB patients suggests that even in the absence of clinical symptoms, ASB can result in significant systemic inflammation, which may contribute to the long-term complications of diabetes, such as diabetic nephropathy, retinopathy, and cardiovascular disease17.
In conclusion, this study highlights the complex interplay between bacterial infections and the inflammatory response in T2DM patients with ASB. The findings underscore the importance of early detection and management of ASB in diabetic patients to prevent exacerbation of chronic inflammation, which may have long-term implications for their health. Further studies are needed to explore the mechanisms underlying the differential immune responses to various pathogens and the long-term effects of ASB on the progression of diabetic complications.
Acknowledgement
The Author thanks the Department of Microbiology, GVPIHCMT for facilitating this work. Author also thanks Dr. P. Sailaja, Dr. R. Poorani and Dr M Krishna Karthik for their support during the study and Mr. N Hanumanth for the statistical analysis.
Conflicts Of Interest
There are no conflicts of interest.
Author Contribution
Designed, performed the experiment and wrote the manuscript: KVSB Vidya Sagar. Analysed and interpreted the data: KVSB Vidya Sagar, P Sarat Jyotsna. Read and approved the final manuscript: P Sarat Jyotsna, Ramnath Karucheri, Narasinga Rao Bandaru.
Funding
The study is a part of Ph.D. thesis of KVSB Vidya Sagar. This is not funded by any agency.
Ethics Statement
Institutional Ethical Committee approval (GVPIHCMT/IEC/20201208/03) was obtained.
Informed Consent
The informed consent was obtained for experimentation from the participants.