Prevalence of Diabetes Mellitus (DM) has been a growing concern across the world with the number of diabetics increasing day-by-day. The world is expected to have 592 million diabetics by 2035 [1]. India has been the “Diabetes Capital” of the world. Diabetic individuals have compromised cell-mediated immunity paving way to repeated infections despite treatment. Urinary Tract Infections (UTI) are quite common and recurrent in DM patients of which a major segment is affected with Asymptomatic Bacteriuria (ASB) [1].

ASBs are usually detected in cases where the clinician investigates for generalized symptoms not pertaining to symptomatic UTI. In majority of DM patients ASB is undetected and un-treated which further progress to complicated acute UTIs that may affect the kidney function on a long run [2]. Currently the treatment of ASB is not

mandate in DM patients as per IDSA 2019 Clinical Practice Guidelines [3]. Hence it is important to study the frequency of occurrence of ASB, the organisms and their antibiotic sensitivity patterns to address the need for effectiveness of treatment to avoid further complications affecting kidney function [2].

Several factors such as metabolic control, duration of diabetes, age and gender of the patients has known to show correlation with ASB occurrence previously [4,5], it still needs to be extensively analysed in the current context as their effect on the DM population and susceptibility to ASB needs to be established.

The current study aims to evaluate the ASB occurrence in DM individuals from northern coastal region of Andhra Pradesh, India and identify the causative organisms for ASB and evaluate their antibiotic sensitivity and resistance patterns that would help the clinicians for planning the accurate prophylaxis.

This laboratory based prospective study was performed in the Microbiology department of GVPIHCMT, Visakhapatnam. Total 258 DM patients of 30-70 years were included in this study. Patients who were immunocompromised, having symptomatic UTIs, on antibiotic treatment, renal obstruction and failure, catheterised patients and pregnant women were excluded. Prior permission was obtained from institutional ethical committee (GVPIHCMT/IEC/20201208/03) and written informed consent was collected from patients before regustering in to the study. Serum and urine samples were collected at the time of enrolment and also after 6 months for the follow-up study to check the recurrence of ASB.

Fasting blood glucose levels, HbA1C, serum creatinine and serum urea were measured in collected blood samples of all the subjects ^[6]. eGFR was calculated using CKD-EPI Creatinine Equation (2021). Urine (mid-stream) samples were collected from participants using standard guidelines. Collected urine samples were inoculated on MacConkey agar, nutrient agar and blood agar and allowed for overnight incubation at 37°C. Detection of organisms was done by following standard antimicrobial procedures. Antibiotic sensitivity for positive organisms was performed using Kerby-Bauer disc diffusion by following CLSI (Clinical Laboratory Standards Institute) 2021 guidelines [7].

Statistical analysis

Data was analysed using SPSS statistical analysis tool and statistical significance was calculated using independent t-test and Mann-Whitney U test. P value <0.05 was considered to be significant.

Study population

Of 258 DM patients, 124 (48.1%) were female and 134 (51.9%) were male. The average age of the participants was 55.95 ± 9.42 years (Table 1).

ASB prevalence

A total of 36% (i.e. 93 patients) of the study population turned positive for ASB. Of the 93 ASB positive patients’ majority were females (61.3%) [Figure 1].

Follow-up of initial ASB positive patients revealed 26% (24 out of 93) had recurrent ASB infection. Females were having higher recurrence of ASB (66.7%) when compared to males (33.3%) [Figure 2].

Bacterial spectrum

Klebsiella was the highly prevalent organism in ASB positive patients (Initial occurrence - 44% and 50% during follow-up study). This was seconded by E. coli (Initial occurrence - 28% and 33% in follow-up), S.aureus had initial occurrence of 22% and 13% during follow-up and the least infective organism was Enterococci with 5% initially and 4% during the follow-up study [Figure 1 & 2].

Antibiotic sensitivity and resistance pattern

Klebsiella was the predominant isolate found in (41 out of 93 ASB positive samples) initially. Maximum sensitivity was observed against Imipenem, Meropenem, Tigecycline (38 of 41 isolates) followed by Amikacin (35 of 41 isolates). All the 41 isolates were found to be resistant to ampicillin, followed by 35 isolates being resistant to Nitrofurantoin. In follow-up for recurrent ASB in the above patients after 6 months, 12 were positive for Klebsiella. Out of 12 isolates, all were sensitive for tigecycline and 11 were sensitive for Amikacin. All isolates were resistant for Ampicillin and 9 were resistant against Nitrofurantoin and Ceftazidime [Table 2].

The second most common ASB causing organism was E. coli (found in 26 of 93) in initial samples. All the isolates were sensitive for Imipenem, Meropenem and Tigecycline. 12 and 10 isolates were resistant to Ampicillin and Ciprofloxacin respectively. In follow-up studies after 6 months 8 patients were found to have recurrent infection with E. coli. All E. coli isolates were sensitive for Imipenem and Meropenem. 4 of the 8 isolates were found to be resistant for levofloxacin [Table 2].

Staphylococcus aureus caused ASB in 21 of 93 patients initially. Of the 21, 20 isolates were characterised to be Methicillin Sensitive Staphylococcus aureus (MSSA) and 1 isolate was Methicillin Resistant Staphylococcus aureus (MRSA). All 20 MSSA isolates were sensitive to cefoxitin, teicoplanin, linezolid. Follow-up post 6months revealed 2 MSSA isolates exhibiting sensitivity against most antibiotics tested except Penicillin, Erythromycin and Cotrimoxazole [Table 2].

The lone MRSA isolate was sensitive to Clindamycin, Erythromycin, Linezolid, Nitrofurantoin, Teicoplanin and resistant to all the other antibiotics tested. 1 MRSA isolate in the follow-up also showed the same sensitivity and resistance pattern as above [Table 2].

Enterococci were the least found isolate causing ASB in 5 of 93 ASB patients initially; which were sensitive to high level Gentamycin, Teicoplanin, Vancomycin and Linezolid. Follow-up post 6 months revealed a lone recurrence of Enterococcal isolate which was sensitive against all the antibiotics tested [Table 2].

Association of HbA1c with ASB

ASB occurrence in DM patients was the highest in individuals with HbA1c level 6.5- 8.0% (63.4% of ASB); Follow-up study in ASB patients also revealed a similar trend with 62.5% patients with recurrent ASB having HbA1c levels between 6.5- 8.0%. ASB occurrence in individuals with controlled HbA1c levels <6.5% was 18.3% during the initial study. Follow-up study after 6 months revealed the recurrence of ASB was the lowest (8.3%) in patients with HbA1c <6.5%. Initial study showed 18.3% of ASB patients were having HbA1c levels >8.0% and this increased to 29.2% of total ASB recurrence in follow-up study [Table 3].

Analysis of baseline characteristics in patient serum

Fasting blood glucose and HbA1c levels

Fasting blood glucose (FBG) levels analysis in the initial and follow-up studies revealed that Klebsiella infection caused a significant increase (157mg/dl to 187mg/dl) in patients with repeated ASB during the follow-up study (Table 4).

The increase in FBG levels was also comparable to the significant increase in HbA1c levels in patient samples positive for Enterococci (7.30% to 8.20%) and Klebsiella (7.30% to 8.31%) indicating poor glycaemic control (Table 4).

Serum creatinine and urea levels

Estimation of serum creatinine levels in initial study revealed that Klebsiella positive ASB patients had significant decrease from 0.92mg/dl to 0.65mg/dl. Whereas in follow-up studies, patient having Enterococci had significantly increased creatinine levels from 0.82mg/dl to 1.31mg/dl. Though these changes are statistically significant all the values are well within the normal range (Table 4). Blood urea levels remained unaffected in ASB and non-ASB patients in both initial and follow-up studies respectively (Table 4).

eGFR levels

In the initial studies, eGFR significantly increased in Klebsiella infected ASB positive patients (89.04ml/min/1.73m² to 113.80ml/min/1.73m²). In follow-up studies, Klebsiella infected ASB positive patients exhibited a similar trend (95.78ml/min/1.73m² to 110.25ml/min/1.73m²). Whereas, in patients infected with Enterococci exhibited significant decrease in eGFR values (95.78ml/min/1.73m² to 67.00ml/min/1.73m²) (Table 4).

DM is a chronic condition with hyperglycaemia due to impaired metabolism caused by both insulin resistance and decreased insulin secretion. Previous studies indicate that diabetics are more prone to frequent UTI infections either symptomatic or asymptomatic.

In the current study, 36% (initial) and 26% (follow-up) were ASB positive of which the majority were females (61.3% initially and 66.7% during the follow-up). These findings coincided with previous literature available [8,9]. In contrast to previous literature [10], Klebsiella was the most common ASB causative organism in the female population 73.17% initially and 75% during the follow-up studies.

Poor glycaemic control (HbA1c 6.5% to 8.0%) individuals, were found to be more susceptible to ASB in both initial (63.4% ASB patients) and follow-up (62.5% ASB patients) studies (Table 3). Poor glycaemic control may also be one of the contributing factors for recurrent ASB which is evident in the data shown in Table 3 and Table 4. Occurrence of ASB was significantly low in patients with good glycaemic control during initial (18.3% of ASB) and follow-up (8.3% of ASB) studies. 

Increase in serum urea and serum creatinine levels are a symptom of progressive kidney damage [11,12]. Significant increase in serum creatinine levels were paralleled with poor glycaemic control (Table 4). Though the increase is well within the normal creatinine range for the short time period, this may play a futuristic role in causing renal damage. Given that the time period of the study is short, blood urea levels were not significantly affected in the overall DM population studied.

Lone case of repeated Enterococcal infection during the follow-up eGFR dropped from 95.78ml/min/1.73m² to 67.00ml/min/1.73m² which may be indicative of onset of compromise in renal function. As a lone case of occurrence this may need further study with a larger population for a longer time period.

Presence of bacteria >10⁵CFU/ml in urine can be a major reason for occurrence of ASB [5]. Of the isolates, gram negative bacteria such as Klebsiella and E. coli were found to be predominant both in the initial and the follow-up studies. Whereas the occurrence of gram-positive bacteria was limited to coagulase negative strains such as Staphylococcus aureus and Enterococci. This is concordance with previous studies available [5,10,13].

Culture sensitivity is important for ASB treatment and influence development of further complications such as recurrent symptomatic UTIs leading to compromise in kidney function with longer duration of diabetes mellitus. Antibiotic sensitivity of the isolated organisms was evaluated using various antibiotics like fluoroquinolones (Ciprofloxacin, Levofloxacin), aminoglycosides (Amikacin, Gentamicin), cephalosporins (Ceftazidime), Nitrofurantoin, Imipenem, Meropenem, Vancomycin and penicillin-G.

Majority of isolates showed high sensitivity to Imipenem, Meropenem, Tigecycline, Amikacin, Linezolid, Teicoplanin, Vancomycin and Clindamycin.  Resistance was noted against antibiotics like Ampicillin, Nitrofurantoin, Ceftazidime, Ciprofloxacin, Levofloxacin and Gentamicin. The finding of other studies also correlated with the present study revealing the similar pattern of antibiotic sensitivity [5,14,15].

Incidence of ASB is becoming more frequent and common among diabetic individuals. This can be linked to several factors such as difference in geographical areas and shifting in causative pathogens with variable drug sensitivity and resistance patterns.  Careful monitoring of glycaemic status may also help to reduce the incidence of ASB. Targeted therapy for ASB based on the antibiotic sensitivity pattern may be of great help to alleviate secondary complications including UTIs and pyelonephritis in diabetic patients.

Acknowledgement

The Author thanks the Department of Microbiology, GVPIHCMT for facilitating this work. Author extends his gratitude to Dr. R. Poorani, Dr. P. Sailaja and Dr. MVS Krishna Karthik for their support. Author also thanks Mr. N Hanumanth, statistician in GVPIHCMT.

Conflict of Interest

There are no conflicts of interest.

Authors' contribution

Designed, performed the experiment and wrote the manuscript: KVSBVS. Analysed and interpreted the data: KVSBVS, RK, PSJ. Read and approved the final manuscript: PSJ, NRB, RK.

Funding

The study is for the Ph.D. thesis of KVSB Vidya Sagar. This is not funded by any agency.

Data availability

Complete set of data and its analysis are included in the manuscript.

Ethics statement

Institutional Ethical Committee approval (GVPIHCMT/IEC/20201208/03) was obtained.

Informed consent

The informed consent was recieved from all the participants.

1. Bharti A, Chawla SP, Kumar S, Kaur S, Soin D, Jindal N, Garg R. Asymptomatic bacteriuria among the patients of type 2 diabetes mellitus. Journal of family medicine and primary care. 2019 Feb 1;8(2):539-43.
2. Meghana BS, Ravi K. Asymptomatic bacteriuria–Spectrum of organisms and antibiotic resistance pattern in women with type 2 diabetes mellitus. APIK Journal of Internal Medicine. 2022 Jul 1;10(3):164-8.
3. Nicolle LE, Gupta K, Bradley SF, Colgan R, DeMuri GP, Drekonja D, Eckert LO, Geerlings SE, Köves B, Hooton TM, Juthani-Mehta M. Clinical practice guideline for the management of asymptomatic bacteriuria: 2019 update by the Infectious Diseases Society of America. Clinical Infectious Diseases. 2019 May 2;68(10):e83-110.
4. Matthiopoulou G, Ioannou P, Mathioudaki A, Papadakis JA, Daraki VN, Pappas A, Souris S, Maraki S, Stathopoulou C, Kofteridis DP. Asymptomatic bacteriuria in patients with type 2 diabetes mellitus. Infectious Disease Reports. 2023 Jan 5;15(1):43-54.
5. Reddy AS, Dasu K, Krishnan V, Mallu RR, Jithendra K, Rao PS. Prevalence of asymptomatic bacteriuria and its antibiotic sensitivity in type-2 diabetic women along the sea coast.
6. Sabiullah M. Estimation of serum creatinine, blood urea nitrogen and urine analysis in patients with diabetes to assess the renal impairments. Int J Adv Biochem Res. 2019;3(2):01-4.
7. Clinical and Laboratory Standards Institute. 2021. Performance standards for antimicrobial susceptibility testing. CLSI supplement M100, 31^st Clinical and Laboratory Standards Institute, Wayne, PA.
8. Venkatesan KD, Chander S, Loganathan K, Victor K. Study on asymptomatic bacteriuria in diabetic patients. International Journal of Contemporary Medical Research 2017;4:480‑3.
9. Vishwanath S, Sarda R, D’souza AO, Mukhopadhyay C. Asymptomatic bacteriuria among patients with diabetes mellitus at a tertiary care center. National Journal of Laboratory Medicine 2013;2:16‑9.
10. Banerjee M, Majumdar M, Kundu PK, Maisnam I, Mukherjee AK. Clinical profile of asymptomatic bacteriuria in type 2 diabetes mellitus: An Eastern India perspective. Indian journal of endocrinology and metabolism. 2019 May 1;23(3):293-7
11. Anjaneyulu M, Chopra K. Quercetin, an antioxidant bioflavonoid, attenuates diabetic nephropathy in rats. Clinical & Experimental Pharmacology & Physiology. 2004; 31:244-248.
12. Shrestha S, Gyawali P, Shrestha R, Poudel, Sigdel M, Regmi P. Serum Urea and Creatinine in Diabetic and nondiabetic subjects. JNAMLS. 2008; 9:11-12.
13. Selvarangan;R. Goluszko;J. Singhal, C.Carnoy, S.Moseley and B.Hudson.Interaction of Dr adhesion with collagen typeiv is a Critical step in E. Coli renal persistence. Infect Immunol 2004;72:4827 4835.
14. Hsueh PR, Hoban DJ, Carmeli Y, Chen SY, Desikan S, Alejandria M, et al. Consensus review of the epidemiology and appropriate antimicrobial therapy of complicated urinary tract infections in Asia-Pacific region. J Infect 2011;63:114-23.
15. Nicole LE. Epidemiology of urinary tract infection. Infect Med 2001;18:153-162.