Abstract

Background: Diabetic patients have been found to be prone to urinary tract infections, and there is a wide gap of information in developing countries regarding the prevalence and antibiotic sensitivity of the pathogens causing this infection. This study was carried out to determine the prevalence, predisposing factors and antibiotic sensitivity of organisms causing urinary tract infections among diabetic patients and non-diabetics in four hospitals in Awka, Anambra State, Nigeria. Method: A total of four hundred and sixty participants (230 diabetic patients and 230 non-diabetics) were enrolled in a cross- sectional study design with 249 males (54.13%) and 211 (45.87 %) females. Clean catch mid-stream urine samples were collected from all participants in sterile containers and analyzed macroscopically and microscopically. Each urine specimen was streaked onto Nutrient agar, MacConkey agar, Cysteine Lactose Electrolyte Deficient agar and Sabouraud’s Dextrose agar, incubated at 37°C for 24h and identified using standard methods. The sensitivity of the isolates to different antibiotics was tested using Kirby-Bauer disc diffusion method. Data obtained were analyzed statistically. Result: The overall prevalence of urinary tract infections among diabetic patients, 63 (27.39%), was significantly higher than that among non-diabetics, 41 (17.83%) (p= 0.014). Gender and previous history of UTI were found to have significant association with urinary tract infection (0.000). Organisms isolated were Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas aeruginosa, Citrobacter spp, Coagulase negative Staphylococcus, Staphylococcus aureus, Enterococcus faecalis, and Candida albicans. The isolates were sensitive to tested antibiotics with Gentamicin (10µg) and Ceftriaxone (30µg) as most effective against Gram negative bacteria isolates while Ampicillin (10µg) and Chloramphenicol (30µg) were most effective against Gram positive bacteria isolates. Conclusion: The prevalence of UTI is significantly higher in diabetics than in non-diabetics with E. coli being the most common isolate.The importance of antibiotic sensitivity testing before treatment is highly recommended.

1. Introduction

Diabetes Mellitus (DM) is a group of metabolic diseases characterized by hyperglycemia, resulting from defects in insulin secretion, insulin action or both ¹. The prevalence of diabetes mellitus has increased over the past decades, and global reports show that it is now approaching epidemic proportions ^{2, 3, 4}. In 2017, the global prevalence of diabetes among adults aged 20-70 years was 8.4% and was responsible for 10.7% of all cases of deaths worldwide ⁵. An estimated 463 million adults aged 20–79 years are living with diabetes ⁴. This represents 9.3% of the world’s population in this age group. The prevalence is expected to rise from 135 million in 1995 to 300 million in the year 2025, 578 million (10.2%) by 2030 and to 700 million (10.9%) by 2045 ⁴. According to WHO ³, the number of people with diabetes in Africa has increased from 4 million in 1980 to 25 million in 2014, and the number is expected to reach 47.1 million by 2045 ⁴. The disease was responsible for more than 366,200 deaths in Africa in 2019 ⁴. The estimated prevalence of diabetes is 2.4% in rural areas, up to 5.9% in urban sub-Sahara Africa ⁴ and between 8-13% in more developed areas such as South Africa ⁶.

Urinary tract infection (UTI) is the most common infection among patients with diabetes mellitus ^{7, 8}, with estimates of diabetics suffering from UTI reaching 10% of patients visiting hospitals ⁹. It is more common among diabetic patients than non-diabetics ¹⁰. Evidence from various epidemiological studies showed that UTI is more common in females with diabetes than in non-diabetic females ^{1, 12, 13}.

The chronic hyperglycemia of diabetes is associated with long term damage, dysfunction, and failure of various organs especially the eyes, genitourinary system, nerves, heart, and blood vessels ¹⁴ and low immunity ¹⁵, and these complications predispose diabetics to urinary tract infections (UTIs) ⁴. Other risk factors associated with increased incidence of urinary tract infections among diabetic patients include low socioeconomic status, advancing age, ¹, sexual intercourse, and type of diabetes mellitus ², metabolic control, and long term complications ¹⁶. UTI in diabetics is associated with a number of serious side effects which include carcinoma of the bladder, gram negative bacteriaemia, sepsis, pyrexia of unknown origin, end point renal failure, hypertension or hypotension, increased prematurity, low birth weight and fetal death ⁶.

Generally, the common etiologic agents of UTI include Escherichia coli, Klebsiella spp., Staphylococcus aureus, Pseudomonas aeruginosa, Enterobacter spp., Enterococcus spp., Proteus spp., Citrobacter spp., Acinetobacter spp. ¹⁷, Staphylococcus saprophyticus, Candida spp. and other pathogenic yeasts ¹⁸. Escherichia coli causes 80-90% of acute uncomplicated bacterial lower tract infections (cystitis) in young women ¹⁷. These agents have also been reported to be associated with UTI in diabetic patients ^{6, 16, 19}.

Antimicrobial agents commonly used in UTI therapy include beta-lactam antibiotics: Ampicillin, Amoxicillin; Fluoroquinolones: Norfloxacin, Ciprofloxacin; third generation Cephalosporins: Cephalexin, Cotrimoxazole, Gentamycin and Nitrofurantoin ^{18, 19}. Uropathogens show wide differences in their susceptibility to these antimicrobial drugs from place to place and time to time ¹⁹. Reports show that diabetic patients using antibiotics experience more intense UTI when compared to those not using the drugs ²⁰. This is as a result of the resistance posed by uncontrolled use of these drugs, thereby exposing them to more serious infections. The aim of this study, therefore, was to determine the prevalence, predisposing factors and antibiotic sensitivity pattern of the organisms causing urinary tract infections among diabetic patients and non-diabetics in four hospitals in Awka, Anambra State, Nigeria.

2. Materials and Methods

The study was a hospital based comparative cross- sectional study conducted from April 2020 to September 2020.

The study was conducted in Awka, the capital city of Anambra State in the South-East geopolitical zone of Nigeria. It covers a land area of about 522 km². Awka has an estimated population of 301,657 as of the 2006 National population census ²¹, and over 2.5million by 2018. Four hospitals, Chukwuemeka Odumegwu Ojukwu University Teaching Hospital (COOUTH), Crest Specialist Hospital, Regina-Caeli Specialist Hospital and Nnamdi Azikiwe University Medical Center, all in Awka South Local Government Area, were used. The study area was an appropriate location as it attends to many patients from the nine towns in the Local Government Area and thus minimizes sample bias due to the wide area covered.

The study population comprised both adult male and female diabetic patients and non-diabetics, aged between 20-80 years, attending both outpatients and inpatients of the selected hospitals, between 8 April 2020 and 30 September 2020.

Socio-demographic data were collected from each study participant using a structured questionnaire. A “clean-catch” midstream urine specimen was collected following the method described by ¹. About 20 ml of urine specimen collected in a sterile, dry, screw capped and wide-mouthed plastic container was labeled with unique sample number, date, and time of collection. The specimens were then transported in ice-pack containers to the laboratory of Applied Microbiology and Brewing, Nnamdi Azikiwe University, Awka, within 2h of collection for further studies ²².

Using a sterile standard calibrated wire loop (0.002ml), a loopful of each urine specimen was directly inoculated onto Nutrient agar, Cysteine-lactose-electrolyte-deficient (CLED) agar and MacConkey agar culture media by streak plate method. The inoculated plates were incubated at 37°C for 24h and examined for bacterial growth. The approximate number of colonies were counted and the number of bacteria (colony forming units (cfu) per millimeter of voided urine) was estimated. A colony count of more than 100,000 (≥10⁵ cfu/ml) was considered a significant bacterial count for positive urinary tract infection ²³. Urine specimens inoculated on Sabouraud Dextrose agar were examined macroscopically for yeast growth after 48h incubation at 30°C. Significant candiduria was determined as urine culture growth ≥10⁴CFU/ml.

A single colony was suspended into Nutrient broth and streaked onto Nutrient agar plates for further identification ²⁴. The inoculated plates were incubated at 37°C for 24h. Pure cultures of bacterial isolates were identified using colony characteristics, Gram reaction and biochemical reactions following standard procedures ²³.The morphological appearance of the fungal isolates were noted as presumptive identification. Following standard procedures, the germ tube test and sugar fermentation tests were carried out on all yeast isolates ²⁵.

Antibiotic sensitivity tests were carried out on pure cultures of bacterial isolates using Kirby-Bauer disc diffusion method ²⁴. The isolates were tested against 13 antibiotics; Ampicillin (AMP) (30µg), Amoxicillin (AMX) (30µg), Ceftriaxone (CEF) (30µg), Ciprofloxacin (CIP) (10µg), Norfloxacin (NOR) (5µg), Nitrofurantoin (NIT) (200µg), Gentamicin (GEN) (10µg), Oxacillin (OX) (10µg), Tetracycline(TET) (10µg), Doxycycline (DOX) (10µg), Chloramphenicol (CHL) (30µg), Erythromycin (ERY) (10µg) and Cotrimoxazole (COT) (25µg). Using sterile wire loop, colonies of each pure culture was suspended in 3ml of physiological saline and the suspension thoroughly mixed. One milliliter of the suspension was transferred into a bijou bottle and diluted with peptone water until the optical density of the suspension matched that of 0.5 McFarland standard solution ²⁵. The standardized test inoculum, (test organism) was inoculated evenly over the entire surface of Muller-Hinton agar (Oxoid), in triplicates using sterile cell spreaders. The antibiotic-impregnated discs were placed on the surface of the culture media using sterile forceps, and after 24h incubation at 37°C, the diameters of the zones of growth inhibition were measured to the nearest whole millimeter using a caliper. The zones of inhibition were interpreted as susceptible (S) or resistant (R) following the ²⁵ guideline.

The data obtained from this study were analyzed using the Statistical Package for the Social Sciences (SPSS) software for windows (version 25). Percentages, frequencies, and cross tabulations were used to summarize descriptive statistics. Pearson Chi-square test was employed to test the existence of association between discrete variables. P-value of <0.05 at 95% confidence interval was considered to indicate statistically significant differences. Odds ratio (OR); Crude Odds Ratio (COR) and Adjusted Odds Ratio (AOR) were used in the analysis. Both bivariate and multivariate logistic regression analyses were employed to ascertain the degree of association between the outcome variable (positive UTI) and independent variables (socio-demographic characteristics and health related risk factors) ²⁶.

3. Results

A total of 460 participants (230 diabetic patients and 230 non-diabetics) were included in the study. Of these, 249(54.13%) were males and 211 (45.87%) females. The participants were aged between 20 to 80 years for both diabetic and non-diabetic participants. The mean age of the diabetic group was 48.8±15.7. One hundred and twenty- three (53.48%) diabetic participants were males while 107 (46.52%) were females. The socio-demographic characteristics of diabetic and non-diabetic patients are as shown in Table 1.

The overall prevalence of UTI was 27.39% among diabetic patients and 17.83% among non-diabetic participants. There was significant difference between the prevalence of UTI among diabetic and non-diabetic participants (P = 0.014). Table 2 shows the prevalence of UTI according to socio-demographic characteristics in diabetic patients and non-diabetics.

In the bivariate analysis, the prevalence of UTI was significantly associated with gender, marital status, place of residence, occupation, and previous history of UTI. No significant association was observed betweenage, educational level, smoking habit and UTI in diabetic patients. Inthe multivariate analysis, the prevalence of UTI was significantly associated with gender and previous history of UTI. The female diabetic patients had higher odds of UTI compared with the males (Table 3). Similarly, diabetic patients with previous history of UTI had higher odds of UTI compared with those without any previous history of UTI (Table 3). However, marital status, place of residence and occupation were not significantly associated with UTI (Table 3).

Sixty-three bacteria and forty-one yeasts were recovered from urine samples of diabetic and non-diabetic participants, respectively. Gram-negative bacteria were the predominant isolates from urine samples of 38 (60.32%) diabetic and 21 (51.22%) non-diabetic participants. Among the diabetic group, eight bacteria and one yeast species were isolated from the urine cultures. The most prevalent bacterial isolates were Escherichia coli 25 (39.68%) while the least isolate was Citrobacter spp. 1 (1.6%). Among the non-diabetics, six bacteria and one fungal species were isolated from urine cultures. The most prevalent was also Escherichia coli 17 (41.46%), while the least was Enterococcus feacalis (2.44%) and Proteus mirabilis (2.44%). The prevalence of organisms isolated from diabetic and non-diabetic participants are presented in Table 4.

Antimicrobial susceptibility patterns of the bacterial isolates from diabetic and non-diabetic participants are presented in Table 5a and Table 5b. Most of the isolates showed 100% sensitivity to Ceftriaxone, Ciprofloxacin, Gentamicin and Chloramphenicol while Escherichia coli, which is the most predominant isolate among the diabetics, showed high level of resistance to Amoxicillin (76%) (Table 5a). Predominant gram-positive isolate, Coagulase negative Staphylococcus (CoNS) showed sensitivity to Ceftriaxone (83.3%), Nitrofurantoin (83.3%), Doxycycline (100%) and Erythromycin (100%) but resisted Ciprofloxacin (50%) and Oxacillin (50%). Staphylococcus aureus isolates also showed 100% sensitivity to Ampicillin, Nitrofurantoin and Chloramphenicol (Table 5b).

4. Discussion

Studies have demonstrated greater susceptibility of diabetics than non-diabetics to urinary tract infections ^{27 12}. The prevalence of urinary tract infections among diabetic and non-diabetic patients, risk factors associated with urinary tract infections in diabetics, spectrum of uropathogens responsible for UTI in diabetic patients and non-diabetics and the sensitivity patterns of the bacterial isolates to antibiotics were investigated.

The findings from this study showed that the overall prevalence of urinary tract infections among diabetic patients (27.4%) was higher than those of non-diabetic patients (17.8%). The high prevalence of UTI among diabetic patients is in line with that of ²⁸, who reported a prevalence of 32.0% among diabetics and 22.0% in non- diabetics. It also agreed with the studies of ^{29, 30}, in which they recorded 25.2% and 33.8% UTI among diabetic patients, respectively. In contrast, ^{2, 24}, reported lower prevalence rates of 19.5% and 13.8%, respectively among diabetic patients. Worku et al. ²⁶, also recorded a low prevalence of 10.9% among diabetics and 4.7% among non-diabetics.

Some other researchers reported higher prevalence of urinary tract infections among diabetic patients and non- diabetics. Prevalence rates of 40% to 50.7% have been reported among diabetics ^{10, 11, 13, 31}. The high prevalence rate could be attributed to emergence of antibiotic resistant bacteria that cause urinary tract infection in diabetics.

There was a significant difference (P < 0.5) in the prevalence of UTI among diabetic patients when compared to non-diabetics and this finding supports the work of many other researchers ^{1, 12, 27, 32}.

Among diabetic patients, a significant difference in the prevalence of UTI was observed among various age groups. A higher prevalence of UTI was observed among age group 40-49 years (54.2%) and age group 50-59 (34.9%) (Table 2). This result agrees with those of ^{33, 34}, who observed a high prevalence in age groups 30-49 years and 51-60 years, respectively. The result obtained is contrary to that of ³⁵, who observed an increase in UTI with increasing order of age. The high prevalence of UTI recorded among the age group 40-49 years could be due to increased rate of sexual activity in this age group.

The incidence of urinary tract infections was found to be significantly higher in female diabetics (43.0%) than in male patients (13.8%) (P = 0.000) (Table 2). This finding is in line with the reports of ^{11, 13}, who reported higher prevalence of UTI among female diabetics. A higher prevalence of UTI was also observed among female non- diabetics (27.9%) than male non-diabetics (9.5%) and this result supports the works of other researchers ^{6, 30, 36}. Contrary to our findings, ³⁷, reported a higher prevalence of UTI in male diabetic patients than in females. The higher prevalence of UTI recorded among female diabetic and non-diabetic patients may be caused by decrease in normal vaginal flora (Lactobacilli), less acidic pH of vaginal surfaces, poor hygienic condition, short and wider urethra, and proximity to the anus ².

Table 3 shows the assessment of the association between various risk factors and UTI in diabetic patients. Age was one of the factors considered and the bivariate logistic regression analysis showed no significant association between age and incidence of UTI in diabetic patients. This finding agrees with the work of ^{24, 30, 38}. In contrast to this result, ³⁹, reported a significant association between age of diabetic patients and UTIs. It was observed (Table 3) that gender and previous history of UTI were associated with high prevalence of UTI in diabetic patients. There was no significant association betweeneducational status, smoking habit of patients and the incidence of UTI in diabetic patients (Table 3). However, ³⁰ reported a significant association between drinking habit, high level of glucose and high prevalence of UTI.

The organisms causing UTI in diabetic patients were similar to those in non-diabetics but with variations in the number of isolates obtained (Table 4). This result corroborates the reports of ⁴⁰, who studied the impact of diabetes mellitus on the spectrum of uropathogens in patients with UTI. The incidence of Pseudomonas aeruginosa and Citrobacter spp. was found only among diabetic patients (Table 4). This observation agrees with the findings by ³⁰. The results (Table 4) also showed that the etiologic agents of UTIs in both diabetics and non- diabetics were mainly bacterial species, and this is in accordance with the report of ³⁶. Most of the isolates from this study belonged to Gram-negative bacteria, with Escherichia coli being the most predominant (40.38%). This finding supports the reports of other researchers ^{6, 10, 22, 37, 41}. The high incidence of Escherichia coli, as suggested by ¹⁰, could be attributed to the fact that they are commensals of the intestines and that infections are most likely to be by faecal contamination due to poor hygiene. The findings, however, disagree with reports by ¹¹ who observed that Coagulase-Negative Staphylococcus was the most prevalent isolate accounting for 37.5%.

Escherichia coli was responsible for 39.7% and 41.5% of urinary tract infections among the diabetic and non-diabetic groups respectively, and this is in line with the reports of ¹². Contrary to our findings, ⁴⁰ reported a prevalence of 67.3% in diabetic patients and 61.8% in non- diabetic group. The lower isolation rate of E. coli in this study when compared with the findings of ⁴⁰, could be attributed to the smaller sample size examined. Klebsiella pneumonia (7.7%) and Proteus mirabilis (4.8%) were the second and third most prevalent bacterial isolates respectively amongst Gram-negative bacteria. The prevalence of Klebsiella pneumoniae among diabetics (7.94%) and non-diabetics (7.32%) supports the reports of ⁴⁰. They isolated 9.3% of Klebsiella pneumoniae among diabetics and 7.3% among non-diabetics. Jagadeeswaran et al. ³⁷, also isolated 8.6% of the organism among diabetics. Obeagu et al. ⁶, however, reported a 31.42% isolation of Klebsiella pneumoniae among diabetics and 28.57% in non-diabetics. Similarly, ¹², also reported the high rate of 20.14% of the organism in diabetics and 19.40% in non-diabetics. The variations in the isolation rates may be as a result of the isolation media used by the researchers.

Table 4 shows a 6.34% isolation of Proteus mirabilis among diabetics and 2.44% in non-diabetics, and the result is comparable with the report of ², who isolated 7.65% of the organism in diabetics. Contrary to our findings, ¹², recorded a 12.23% of the organism in diabetics and 10.44% in non-diabetics, while ³⁰, had 9.3% isolation in diabetics.

As presented in Table 4, Coagulase Negative Staphylococcus (18.27%) and Staphylococcus aureus (17.31%) were the second and third most prevalent Gram positive bacterial isolates, respectively. This result supports the findings of ²⁴ who observed CoNS (24.2%) and Staphylococcus aureus (18.2%) to be the second and third most common isolates, respectively. Woldemariam et al. ⁴², also reported Coagulase Negative Staphylococcus as the second most isolated bacteria after E. coli. In contrast, the second most common isolate in the report by ³⁶, was Enterococcus feacalis (10.9%). The isolation rate of Coagulase Negative Staphylococcus from diabetic patients (19.04%) and non-diabetics (17.07%) is as presented in Table 4. This result is comparable with that of ³⁰. Gram-positive bacteria are not common uropathogens ², however, due to contamination, they have been found to always invade the urinary tract infection and cause UTI ⁴³. The prevalence of Staphylococcus aureus among diabetics (14.29%) and non-diabetics (21.95%) (Table 4), is higher than that (0.8% in diabetics and 0.6% in non- diabetics) reported by ⁴⁰. The least isolated gram positive bacteria in this study were Enterococcus feacalis, with a prevalence rate of 3.17% in diabetics and 2.44% in non-diabetics. This result agrees with that found by ³. However, much higher results of 9.3% and 16.2% were reported by ³⁰ and ⁴² respectively.

Yeasts, particularly Candida spp. are a common predisposing factor of UTI in diabetes mellitus patients ⁴³. Candida albicans isolated from diabetics (3.17%) and non-diabetics (7.32%) are shown in Table 4. The results obtained are comparable with 2.2% in diabetics reported by ³⁰. However, higher rate of 17.9% was reported by ⁴². Mogaka et al. ³⁹, reported an increase in resistance of isolated bacterial organisms to available antibiotics among diabetic patients. Tables 5a and Table 5b show that, a number of bacterial isolates from diabetic and non- diabetic patients were sensitive to the tested antibiotics. Most of the gram negative bacterial isolates from diabetic patients were sensitive to Beta-lactams, Quinolones and Aminoglycosides (Table 5a). Among the antibiotics tested, Gentamicin (10µg) and Ceftriaxone (30µg) were found to be the most effective drugs against gram negative bacterial isolates. While this finding agrees with the report of ², it disagrees with that of ⁴⁴, who observed quinolones to be the most effective agents against isolated gram negative bacilli. This variation in susceptibility may be due to changing trends of antimicrobial susceptibility pattern of these urinary tract pathogens from place to place ³⁶.

In the present study, Tetracycline (10μg), Ampicillin (30μg) and Amoxicillin(30μg) were the least effective antimicrobials against gram negative bacterial isolates as most of the organisms showed high level of resistance to the antibiotics.

Most of the isolated gram positive organisms were susceptible to the antibiotics tested (Table 5b). Ampicillin and Chloramphenicol were found to be the most effective drugs against gram positive bacterial isolates. While ³⁹, recorded 50.0% of resistance to Ampicillin and Ciprofloxacin, ², reported a lower resistance to Ampicillin and Ciprofloxacin (20.0%), which is supported by the result obtained in this study.

5. Conclusions

The prevalence of UTI was significantly higher in diabetics than in non-diabetics. Gender and previous history of UTI were observed to be risk factors of UTI among diabetic patients. Escherichia coli was observed to be the most prevalent bacterial isolate. Routine urine cultures and antibiotic sensitivity testing of samples from patients is indispensable in the prevention and treatment of UTI.

Ethical Consideration

Ethical approval was obtained from the Ethical Research Committee of Chukwuemeka Odimegwu Ojukwu University Teaching Hospital, Awka before conducting the study.

References