Bacterial association in urinary tract infection and their drug resistance among patients in Rajshahi, Bangladesh

INTRODUCTION

Urinary tract infection (UTI) is an infection in any part of the urinary systems such as kidney, ureters, bladder, and urethra. It can be classified as the upper tract infection or the infection of the kidneys (pyelonephritis) and lower tract infection or the infection of the bladder (cystitis), but the infection of the urethra and prostate are also considered as the lower UTIs. This infection can be asymptomatic, acute, chronic, and complicated, or uncomplicated according to how they occur along the urinary tract.¹

Over time, UTI has become a severe public health concern worldwide. UTI that is caused by antibiotic-resistant bacteria can make the treatment difficult and can lead to complications.^2,3 According to the most recent global epidemiological data of UTIs, around 150 million people globally suffer from UTI every year. Women in the age range 16-64 are more likely to get UTIs and one in every five women globally suffers from UTI at least one time in their life. Women are more affected by UTI than men because of their physical structure though men over the age of 60 years with prostatic hypertrophy are at a greater risk.⁴ Some factors such as gender, age, race, circumcision, human immunodeficiency virus (HIV), diabetes, urinary catheter, genitourinary tract abnormalities, and pregnancy are the major risk factors for UTIs.^1,5-13

UTIs can be community-acquired or nosocomial. According to recent data, the prevalence of community-associated UTI (CAUTI) is 17% with major risk factors are age, history of UTI, sexual activity, and diabetes. However, the occurrence of hospital-associated UTI (HAUTI) is 12.9, 19.6, and 24% in the United States, Europe, and developing countries, respectively.¹⁴ Although E. coli is the most predominant microorganism in UTI, some other pathogens include Klebsiella, Enterococcus, Staphylococcus, Pseudomonas, Proteus, Streptococcus, Acinetobacter, Citrobacter and Candida.^15-17

Bangladesh is a densely populated country and most of the people in this country living in a condition where people get affected by various bacterial infections due to lack of knowledge, and unhygienic lifestyle. According to a recent study in Bangladesh, pathogens responsible for UTI acquired resistance to antibiotics. Scientists warn that there could be a higher risk of drug resistance if antibiotics are prescribed without sensitivity or laboratory culture tests. Taking the recent scenario regarding drug resistance into consideration, scientists anticipated that death will rise from 7, 000, 00 (current death) to 10 million people per year by the year 2050 due to antibiotic-resistant bacterial infections.¹⁸ Therefore, our study was designed to detect the prevalence, causative agents, and their antimicrobial susceptibility pattern among UTI patients over a period of 23 months. As drug resistance pattern among antibiotics varies with their selective use in treatment within different localities, here goes the aspiration of our effort to take a depiction of bacterial antibiotic sensitivity in our region.

METHODS

Study area, period, and population

This study was carried out in the microbiology laboratory of Jazeera diagnostic center which is located in Kajihata, Lakhmipur, Rajshahi city, Bangladesh. About 1,760 UTI suspected patients were included to conduct this study and the duration of this study was from January 2019 to November 2020 (a total of 23 months).

Collection of samples

In brief, midstream urine samples were collected in a wide-necked screw-top sterile container, and then, all the containers were labelled with the date, time, and name of the patients. Sample processing was performed within 2 hours of sample collection and in certain cases, they were kept at 4°C for less than 24 hours.

Process of culturing urine samples

To culture, 0.1 ml urine sample was spread on the surface of sterile nutrient agar (NA), and MacConkey agar (MAC) plates (60×15 mm) following standard bacteriological technique. After inoculation, all the plates were incubated aerobically at 37°C for 18-24 hours and following incubation, all the plates were examined in terms of the number of colonies and the colony morphology. The samples were considered positive when the colony number was ≥10⁵ colony forming unit/ml. Colony characteristics of those isolates were marked and recorded.

Characterization of isolates

To obtain pure culture, colonies were picked up from agar media and sub-cultured. Gram staining and biochemical tests were also performed to identify the isolates as per the standard protocol followed in the microbiology laboratory.¹⁹ Antibiotic susceptibility tests were performed following the identification of the pure culture isolates.

Antibiotic susceptibility test

The modified Kirby-Bauer disc diffusion method was followed to observe the sensitivity pattern of the isolates. In brief, isolated colonies were spread over Muller-Hinton Agar media, and then, paper discs of antibiotics were placed on top of it. Following incubation at 37°C for 24 hours, the zone of inhibitions (ZOIs) were measured around the colonies to detect their susceptibility or resistance by comparing with the clinical and laboratory standard institute (CLSI) guideline.²⁰ However, antibiograms were performed against a total of 14 antibiotics namely amikacin (30 mcg), cefixime (5 mcg), imipenem (10 mcg), meropenem (10 mcg), nalidixic acid (50 mcg), nitrofurantoin (300 mcg), gentamicin (10 mcg), co-trimoxazole (25 mcg), amoxiclav (25 mcg), cefuroxime (30 mcg), ceftriaxone (30 mcg), ceftazidime (30 mcg), ciprofloxacin (5 mcg), and levofloxacin (5 mcg).

RESULTS

Distribution of positive and negative samples

Figure 1A shows the number of positive and negative samples from January 2019 to November 2020. Among 846 suspected patients, a total of 242 infected and 604 uninfected patients were found in the year 2019. Moreover, the number of infected and uninfected patients in the year 2020 remained 285 and 629 out of 914 total assumed patients, respectively.

However, there was no significant consistency observed in the increase or decrease of infected patients’ number month-wise among 23 months study period. The highest number of infected patients was found in August 2019 (30) and in July 2020 (45). Conversely, the lowest number of infected patients was received in April 2019 (13) and January 2020 (18).

In our study, it was observed that almost 70% of samples were found as negative (1233 samples) and 30% of samples were positive (527 samples) throughout the study period (Figure 1B).

Age- and gender-wise distribution of positive samples

The highest infection was observed in the age group 31-40 and 21-30 for males and females, respectively (Figure 2A). However, the incidence of infection slowly declined in both males and females following the age range. Our data demonstrated that among all the positive samples, 65.28% (344) were female and 34.72% (183) were male patients (Figure 2B).

Distribution of microorganisms among the positive samples

Our data revealed that E. coli was responsible for most of the urinary tract infections. As the most predominant organism, E. coli was approximately 50.09% followed by Klebsiella spp. (30.55%), Pseudomonas spp. (18.02%), Enterococcus spp. (0.379%), and Citrobacter spp. (0.948%) (Table 1).

During our study period, we did not observe any multiple positive urine cultures from any patient.

Antibiotic sensitivity test

A degree of sensitivity/resistance was observed against E. coli, Klebsiella spp., and Pseudomonas spp. The lowest sensitivity was observed against Amoxiclav by both E. coli (15.91%) and Pseudomonas spp. (16.84%), where Klebsiella spp. showed the lowest 25.47% sensitivity against co-trimoxazole. However, 0% sensitivity was observed against nalidixic acid and amoxiclav by Enterococcus spp.; and against amikacin, cefixime, nalidixic acid, co-trimoxazole, amoxiclav, and ceftazidime by Citrobacter spp. Except for Pseudomonas spp. that showed the highest sensitivity against meropenem, all the other isolates showed the highest sensitivity against nitrofurantoin. However, Enterococcus spp. and Citrobacter spp. showed 100% sensitivity against the three antibiotics namely imipenem, meropenem, nitrofurantoin. Overall, the highest resistance was observed against amoxiclav and the highest sensitivity was detected against nitrofurantoin (Table 2). Nevertheless, our data revealed a scary scenario where only 5 bacterial isolates showed resistance to only one antibiotic, 17 isolates showed resistance to two antibiotics, 305 isolates showed resistance to 3 antibiotics and 200 bacterial isolates showed resistance to 4 or more than 4 antibiotics (Figure 3).

DISCUSSION

Urinary tract infection has reached a critical stage where people in the USA had to expense about 3.5 billion dollars per year on treatment but the scenario is even worse in developing countries like Bangladesh. In the USA, about 2.8 million bacterial infections have acquired antibiotic resistance per year.²¹ Drug resistance has made this infection more complicated. According to some recent research, most of the etiological agents of UTI are becoming resistant to several antibiotics such as Ampicillin which was largely used against UTI.²² Our study aimed to evaluate the predominating microorganisms present in UTI infection and antibiotic susceptibility pattern of the isolated bacteria. To conduct this study, a total of 1760 suspected patients’ samples were tested where 70% of samples did not show any incidence of UTI.

Gram-negative organisms that in general reside in the gut are more responsible for UTI and in most cases, E. coli is accountable for 80-90% of primary infection.²³ Our study highlighted E. coli as the highest predominant organism with 50.09% prevalence which is comparable to other studies where 40-46% of UTI infections were caused by E. coli.^24-27 In our study, Klebsiella spp. was found as the second highest predominating organism (30.55%) followed by Pseudomonas spp. (18.02%), Enterococcus spp. (0.379%), and Citrobacter spp. (0.948%).

Out of suspected patients of UTI, 30% were found infected where 70% did not show any significant infection which very closely correlates with a previous study where 31% samples were found positive for UTI.²⁸ Nonetheless, the highest rate of infection was observed among the females (65.28%) aged 21-40. A higher incidence of UTI infection in females than males was very commonly observed in the earlier investigations and the prevalence is due to their physical structure where the anus is close to the vagina in females in association with the vaginal colonization with uropathogens, sexual activity, pregnancy and obstruction.^28,29-34 Conversely, in men, the reasons behind UTI may include insertive anal intercourse or lack of circumcision or having sexual partner with vaginal colonization with uropathogenic microorganisms or lack of immunity.^{35, 36}

In our study, we observed a wide extent of resistance of uropathogens against several antibiotics which is a matter of great concern. The most predominating E. coli showed 95.45% sensitivity to Nitrofurantoin and 84.09% resistance to Amoxiclav. Moreover, most of the uropathogens of this study including E. coli, Pseudomonas spp., Enterococcus spp., and Citrobacter spp. showed the highest resistance to amoxiclav which is being used as a first-line treatment for lower UTIs. Our result also revealed that 95.8% isolates were resistant to 3 or more than 3 antibiotics which we may call multi-drug resistant (MDR) which is comparable with an earlier report.³⁷ A recent research in 2019 claimed that Bangladesh, along with some of its neighboring countries, is responsibly harboring drug-resistant microbes that might play a role in disseminating them throughout the world by humans, animals, agricultural products, and water. This research addresses significant gaps in surveillance, susceptibility testing methods, their interpretation that is creating doubt in methodologies and therefore, making it difficult to compare with other studies.³⁸

CONCLUSION

Antibiotic resistance can be reduced by conducting such antibiotic surveillance studies, creating awareness in public and by concerted efforts between doctors, scientists, students, pharmaceutical industries, and policymakers. Changing trends in the antibiotic sensitivity profiles of the isolates need to be monitored as there is limited availability of newer drugs and the emergence of resistant bacteria far exceeds the rate of new drug development.

ACKNOWLEDGEMENTS

The authors hereby express their deep gratitude to the Jazeera diagnostic center, Kajihata, Lakhmipur, Rajshahi, Bangladesh for the technical supports provided throughout the study period.

Funding: No funding sources

Conflict of interest: None declared

Ethical approval: The study was approved by the Institutional Ethics Committee

REFERENCES

1. 1. Odoki M, Aliero AA, Tibyangye J, Maniga JN, Wampande E, Kato CD, et al. Prevalence of bacterial urinary tract infection and associated factors among patients attending hospitals in Bushenyi district, Uganda. Int J Microbiol. 2019;2019:8.
2. 2. Foxman B. Epidemiology of urinary tract infections: incidence, morbidity, and economic costs. Dis Mon. 2003;49(2):53-70.
3. 3. Petersen I, Hayward AC. Antibacterial prescribing in primary care. J Antimicrob Chemother. 2007;60(1):43-7.
4. 4. Parveen R, Rahim I. Study of Bacterial Pathogens in Urinary Tract Infection and their Antimicrobial Sensitivity Pattern. Bangladesh J Infect Dis. 2017;4(2):40-4.
5. 5. Conway PH, Cnaan A, Zaoutis T, Henry BV, Grundmeier RW, Keren R. Recurrent urinary tract infections in children: risk factors and association with prophylactic antimicrobials. JAMA. 2007;298(2):179-86.
6. 6. Dias CS, Silva JM, Diniz JS, Lima EM, Marciano RC, Lana LG, et al. Risk factors for recurrent urinary tract infections in a cohort of patients with primary vesicoureteral reflux. Pediatr Infect Dis J. 2010;29(2):139-44.
7. 7. Banu A, Jyothi R. Asymptomatic bacteriuria in HIV positive individuals in a tertiary care hospital. J HIV Hum Reprod. 2013;1(2):54.
8. 8. Iduoriyekemwen NJ, Sadoh WE, Sadoh AE. Asymptomatic bacteriuria in HIV positive Nigerian children. J Med Biomed Res. 2012;11(1):88-94.
9. 9. Ibadin M, Onunu A, Ukoh G. Urinary tract infection in adolescent/young adult Nigerians with acquired human immuno deficiency disease in Benin city. J Biomed Sci. 2006;5(2):55-60.
10. 10. Mladenović J, Veljović M, Udovicić I, Lazić S, Segrt Z, Ristić P, et al. Catheter-associated urinary tract infection in a surgical intensive care unit. Vojnosanit Pregl. 2015;72(10):883-888.
11. 11. Yuyun MF, Angwafo III FF, Koulla-Shiro S, Zoung-Kanyi J. Urinary tract infections and genitourinary abnormalities in Cameroonian men. Trop Med Int Health. 2004;9(4):520-5.
12. 12. Nicolle LE. Uncomplicated urinary tract infection in adults including uncomplicated pyelonephritis. Urol Clin North Am. 2008;35(1):1-12.
13. 13. Nelson JM, Good E. Urinary tract infections and asymptomatic bacteriuria in older adults. Nurse Pract. 2015;40(8):43-8.
14. 14. Tandogdu Z, Wagenlehner FM. Global epidemiology of urinary tract infections. Curr Opin Infect Dis. 2016;29(1):73-9.
15. 15. Ronald A. The etiology of urinary tract infection: traditional and emerging pathogens. Dis Mon. 2003;49(2):71-82.
16. 16. Aswani SM, Chandrashekar U, Shivashankara K, Pruthvi B. Clinical profile of urinary tract infections in diabetics and non-diabetics. Australas Med J. 2014;7(1):29-34.
17. 17. Rizvi M, Khan F, Shukla I, Malik A, Shaheen. Rising prevalence of antimicrobial resistance in urinary tract infections during pregnancy: necessity for exploring newer treatment options. J Lab Physicians. 2011;3(2):98-103.
18. 18. Moue A, Aktaruzzaman SA, Ferdous N, Karim MR, Khalil MM, Das A. Prevalence of urinary tract infection in both outpatient department and in patient department at a medical college setting of Bangladesh. Int J Biosci. 2015;7(5):146-52.
19. 19. Collee JG, Fraser AG, Marmion BP, Simmons A. Tests for identification of Bacteria. In: Mackie and McCartney Practical Medical Microbiology. 14th ed. London: Churchill Livingstone. 1996;131-49.
20. 20. Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing. Twenty-Sixth Informational Supplement. CLSI document M100-S26. Wayne (PA): CLSI. 2016;50-6.
21. 21. Flores-Mireles AL, Walker JN, Caparon M, Hultgren SJ. Urinary tract infections: epidemiology, mechanisms of infection and treatment options. Nat Rev Microbiol. 2015;13(5):269-84.
22. 22. Rosana Y, Billy M, Ocviyanti D. In vitro resistance pattern of urinary tract infections-causing bacteria to ampicillin and ciprofloxacin. Obstet Gynecol Int J. 2019;10(5):372-6.
23. 23. Katouli M. Population structure of gut Escherichia coli and its role in development of extra-intestinal infections. Iran J Microbiol. 2010;2(2):59-72.
24. 24. Kayima JK, Otieno LS, Twahir A, Njenga E. Asymptomatic bacteriuria among diabetics attending Kenyatta National Hospital. East Afr Med J. 1996;73(8):524-6.
25. 25. Moges AF, Genetu A, Mengistu G. Antibiotic sensitivities of common bacterial pathogens in urinary tract infections at Gondar Hospital, Ethiopia. East Afr Med J. 2002;79(3):140-2.
26. 26. Wanyama J. Prevalence, bacteriology and microbial sensitivity patterns among pregnant women with clinically diagnosed urinary tract infections in Mulago Hospital Labour Ward. Makerere University, Kampala, Uganda, 2003, M.Ed. dissertation of Wanyama. 2003.
27. 27. Mayanja R. The Prevalence of Asymptomatic Bacteriuria and Associated Factors among Women Attending Antenatal Clinics in Lower Mulago Hospital, Uganda. ASRJETS. 2016;25(1):131-48.
28. 28. Majumder MM, Ahmed T, Ahmed S, Khan A, Saha C. Antibiotic Resistance in Urinary Tract Infection in a Tertiary Care Hospital in Bangladesh-A Follow-up Study. Med Today. 2019;31(1),9-14.
29. 29. Sewify M, Nair S, Warsame S, Murad M, Alhubail A, Behbehani K, et al. Prevalence of Urinary Tract Infection and Antimicrobial Susceptibility among Diabetic Patients with Controlled and Uncontrolled Glycemia in Kuwait. J Diabetes Res. 2016;7.
30. 30. Al Benwan K, Al Sweih N, Rotimi VO. Etiology and antibiotic susceptibility patterns of community- and hospital-acquired urinary tract infections in a general hospital in Kuwait. Med Princ Pract. 2010;19(6):440-6.
31. 31. Ebie M, Kandakai-Olukemi YT, Ayanbadejo J, Tanyigna KB. Urinary tract infection in a Nigeria Military Hospital. Nig J Microbiol. 2001;15(1):31-7.
32. 32. Kumar MS, Lakshmi V, Rajagopalan R. Related Articles, Occurrence of extended spectrum beta-lactamases among Enterobacteriaceae spp. isolated at a tertiary care institute. Indian J Med Microbiol. 2006;24(3):208-11.
33. 33. Khan AU, Musharraf A. Plasmid mediated multiple antibiotic resistance in P. mirabilis isolated from the UTI patients. Medical Sci Mon. 2004;10(11):598-602.
34. 34. Stamm WE. Urinary Tract Infection and Pylonepritis, In Harrisons Principles of Internal medicine, ed. D. L. Kasper et al., 16th Edition (McGraw Medical Publishing Division, New York, 2005). 1715.
35. 35. Barnes R, Roddy R, Daifuku R, Stamm W. Urinary tract infection in sexually active homosexual men. Lancet. 1986;327(8474):171-3.
36. 36. Rosenberger WF. Circumcision and urinary tract infection. JAMA. 1992;268(1):55.
37. 37. Badri AM, Mohamed SG. Clinical Epidemiology and Antibiogram of UTI Patients Attended Different Hospital in Khartoum, Sudan. Clin Microbiol. 2017;6(5):301.
38. 38. Ahmed I, Rabbi MB, Sultana S. Antibiotic resistance in Bangladesh: A systematic review. Int J Infect Dis. 2019;80:54-61.