Uropathogens and their antimicrobial susceptibility pattern: A retrospective study in a district level hospital in Western Nepal [version 1; peer review: 2 approved with reservations, 1 not approved]

Background: Urinary tract infection (UTI) is a common cause of hospital visits. There is an increasing trend of resistance of uropathogens to antibiotics worldwide. The aim of this study was to identify the common uropathogens, along with their antimicrobial susceptibility. Methods: This retrospective cross-sectional study was conducted from April 2018 to April 2020 at Beni hospital. All patients with urinary tract infection visiting Beni hospital during this time and who had urine culture sensitivity tests done were included in this study. Urine samples were first cultured on cystine lactose electrolyte-deficient agar by a semi-quantitative technique, and then incubated aerobically for 18–24 h at 37 °C. The identified bacterial isolates were tested for antimicrobial susceptibility by the Kirby–Bauer disc diffusion technique. Results: Of the 1173 samples, 164 (14%) samples showed significant growth. Escherichia coli (74%) was the most common causative organism. E. coli was sensitive in 113 cases (95%) out of 119. Amikacin was tested in 87 isolates that showed 99% sensitivity. Other commonly used antimicrobial agents had lower sensitivity rates: gentamicin (83%), ciprofloxacin (75%), ceftriaxone (59%), cefixime (56%), cotrimoxazole (55%), cefotaxime (41%), and ampicillin (38%). Conclusions: E.coli is the most common pathogen associated with urinary tract infection. Nitrofurantoin and amikacin can be good empirical agents for treating UTI in patients coming to Beni hospital. Open Peer Review


Introduction
Urinary tract infection (UTI) is one of the common health problems affecting people of all ages, race/ethnicity, sex, and circumcision status. It is caused mostly by bacteria, but viruses and fungi have also been implicated in rare cases. 1,2 Several studies have reported that there is an increased resistance of uropathogens to a number of common broadspectrum antibiotics worldwide. [3][4][5] As the resistance patterns differ across different areas, every hospital should formulate their own anti-microbiogram for common infections, so as to guide the appropriate treatment.
The objective of this study is to find the prevalence of common uropathogens and, secondly, to identify antimicrobial sensitivity and resistance patterns to those pathogens.

Methods
Study setting, design, population and sampling techniques A retrospective cross-sectional study was carried out in Beni hospital. A convenient sampling technique was used and all patients with urinary tract infection visiting Beni hospital from April 2018 to April 2020 and had urine culture sensitivity tests done were included in this study.
Urine sample collection 15-30 mL of urine was collected in a sterile leak-proof urine container. A midstream clean-catch specimen was taken. Patients were first asked to cleanse the urethral area before collecting the specimen. Specimens received in the laboratory were processed within 2 hours. Transport medium for urine specimens, such as 1.8% boric acid, sodium chloride or polyvinylpyrolidine, was used.

Culture, identification technique and antimicrobial susceptibility testing
The sample received was inoculated in the cysteine lactose electrolyte deficient agar media with a 1-mL calibrated loop of internal diameter, 0.001 mL volume of urine specimen using a semi-quantitative method. After inoculation, it was incubated at 37°C overnight for visible growth. A growth of >10 5 colony forming unit/mL was considered as significant bacteriuria. Bacterial identification was done using standard bacteriological techniques. The antibiotic susceptibility tests of the isolates against different antibiotics were done using Mueller Hinton agar (MHA) by the standard disk diffusion technique of modified Kirby-Bauer method as recommended by the Clinical & Laboratory Standards Institute. In this study, if the isolates were resistant to at least one agent in three or more antimicrobial categories, they were regarded as multi-drug resistant (MDR) organisms.

Potential biases
This study is subject to selection bias as samples were taken conveniently and confirmation bias as observer might be familiar with common drugs that are resistant to pathogens Data processing and analysis Data entry and analysis were done by using SPSS version 25.0 (RRID:SCR_019096); JASP (RRID:SCR_015823) is an open-source alternative to SPSS. Descriptive statistical methods were carried out for data analysis.

Ethical consideration
Ethical clearance was obtained from the national ethical review board, Nepal Health Research Council. The reference number of the ethical letter was 1886 dated 20 th January, 2021.

Consent
Hospital data were reviewed, and patients were not directly involved in the research; approval from the hospital was taken and consent from the patient had been waived by the Nepal Health Research Council

Results
Urine samples from 1173 symptomatic patients were received for urine culture during the study period. Out of 1173 samples, 164 urine samples (14%) showed significant growth of at least one of the uropathogens tested in this study: E. coli, Staphylococcus aureus, Klebsiella spp., Acinetobacter spp. The most common pathogen isolated was E. coli (74%) followed by Klebsiella spp. as shown in Table 1.

Discussion
As with other infections, UTIs are managed initially with empirical antibiotics till the urine culture reports become available. In Nepal, most hospitals in rural areas do not have proper microbiology laboratories performing bacterial culture, so treatment is solely empirical in such cases. Our study showed E. coli as the most common pathogen (74%) causing UTI, followed by Klebsiella spp. (13%) and Acinetobacter spp. (10%). Similar findings have also been reported in different studies conducted in Nepal and other countries. [4][5][6][7] So, empirical antibiotics to treat UTI should target E. coli.
Among 119 samples of E. coli tested for nitrofurantoin, it was sensitive in 113 cases (95%). Amikacin was tested in 87 isolates that showed 99% sensitivity. Other commonly used antimicrobial agents had lower sensitivity rates: gentamicin (83%), ciprofloxacin (75%), ceftriaxone (59%), cefixime (56%), cotrimoxazole (55%), cefotaxime (41%), and ampicillin (38%). Various studies done in Nepal have also shown lower sensitivity of the pathogen to these agents. 3,14 Our study suggests that nitrofurantoin would be a good first-choice oral antibiotic for managing lower UTI such as cystitis. However, in pyelonephritis or prostatitis, nitrofurantoin is not recommended as it does not attain sufficient concentration in these tissues. Based on our study results, aminoglycoside amikacin (parenteral) can be used empirically for pyelonephritis, or when a patient does not tolerate oral medicine. In a prospective cohort study conducted in Singapore from 2015 to 2016, E. coli was sensitive to amikacin in 100% of the cases. 6 Other commonly used oral antimicrobial agents like ciprofloxacin, cotrimoxazole and cefixime had lower sensitivity to E. coli. They had been used rampantly in Nepal in the past, mainly for typhoid fever. This explains the increase in resistance to these agents. Several studies done in Nepal and abroad have also shown higher resistance of E. coli to these antibiotics. 3,4,7-10 Ceftriaxone one of the most commonly used antibiotic in hospitalized patients in Nepal, however its sensitivity to E. coli was only 59%, hence its empirical use in hospitalized UTI patients should be discouraged.
Klebsellia spp. was found to be more sensitive to antimicrobrial agents as compared with E. coli. It was sensitive in all 16  Multidrug-resistant organisms are resistant to at least one agent in three or more classes of antimicrobial agents. The rising incidence of a multidrug resistance phenotype of extended-spectrum beta-lactamase (ESBL) genes and fluoroquinolones resistance, has become a global concern because of their potential cause of serious infections which are difficult to treat. 12 In this study, MDR was isolated in 52 out of 122 cases of E. coli (43%), 13 out of 16 cases of Acinetobacter spp. (81%) and five out of 21 cases of Klebsiella spp. (24%). Significant proportions of MDR uropathogens were seen in other studies too, done in different hospitals of Nepal. 5,13 Antimicrobial resistance (AMR) is a major concern in both developed and developing countries as various studies have shown its rising incidence. It has posed a major challenge for successful treatment of infectious diseases. With increased prevalence of irrational and injudicious use of antimicrobial agents and inadequate antibiotic stewardship programs, it is a major burden for Nepal. 14 Even in rural areas like Beni, Myagdi, all classes of antimicrobial agents are easily available. In our experience antibiotics have been sold mostly without proper diagnostic evaluation of patients and prescription of physicians in both major cities and rural areas of Nepal.
In Beni hospital, culture and sensitivity were not routinely sent in all clinically suspicious cases of urinary tract infections.
Also, sensitivity was not tested on all appropriate classes of antimicrobial agents. Had it been done, we would have a broader view of sensitivity and resistance pattern. As we are collecting more data on this matter, we will have analysis of more data in the future which would give a more accurate prospect of the antimicrobial susceptibility pattern in this hospital.
The susceptibility patterns of antimicrobial agents to microbes vary from country to country and also in different regions of the same country. The guidelines used in Western countries may not be useful in Nepal. It is necessary to identify the sensitivity pattern in a particular location and to develop the treatment protocol accordingly. Very few studies have been published regarding AMR in the Gandaki province of Nepal. 14 This study will help for selection of appropriate empirical antimicrobial agents for treatment of UTI in this region.
The limitation of the study is external validity. As data were collected from a single hospital, our findings cannot be generalized and multicenter studies with larger sample size are needed to find out the real scenario of antimicrobial resistance pattern and formulation of treatment guidelines accordingly. Also patient information like genitourinary malformations, prior exposure to antibiotics, recent hospitalization or prior history of UTI were not taken into consideration, which can be important risk factors for resistant uropathogens.
Conclusions E. coli is the most common pathogen associated with urinary tract infection in Beni hospital. It is resistant to broadspectrum penicillin, third-generation cephalosporins and fluoroquinolones. Resistance to nitrofurantoin is low and could be the antibiotic of choice for uncomplicated cystitis. Amikacin showed promise as a suitable intravenous agent but needs further studies with adequate sample size

Open Peer Review Introduction
In the Introduction section, the authors need to show the importance of performing local antimicrobial susceptibility data, e.g elaborate with WHO surveillance program (GLASS) as the source of information. This is an important point to strengthen the rationale of the article.

Methods
In the Methods section, information regarding microbiology procedures has been written properly. However, the authors need to add more about the study population, data collection, and variable definition, for example: (1) Describe the site of data collection, e.g Beni Hospital; ○ (2) How do the authors collect the data retrospectively, e.g using medical records and/or laboratory information systems? If both of them are used as the source of the data, then how do the authors elaborate on both of the data? Please clarify and explain in the Methods section; ○ (3) Lastly, the authors need to define the definition of a patient with a UTI in this study.

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The important information that is also needed is the antibiotic that is used in this study. This needs to be explained and described clearly in the Methods section, as well as the quality control of the AST procedure.
○ Please indicate and clarify that no technical method and/or supplies (e.g reagent, ○ instrument, etc) was changed between 2018 -2020.
Please provide references in the Methods section, as well as the version of CLSI.

Results
In the Results section, there is the statement: "out of 1173 samples, 164 urine samples (14%) showed significant growth of at least one of the uropathogens tested in this study: E. coli, Staphylococcus aureus, Klebsiella spp., Acinetobacter spp." This might be better to provide information among 1173 specimens; how many urine specimens have shown growth of the organism? This is confusing since only 164 were included from 1173, which indicates almost half of the population was rejected. Please clarify and write more information about this in the results.

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The authors need to clarify the purpose for tabulating between gender and certain isolate and might be more informative if the data are reported separately between years (looking for trends).
© 2021 Rhajbhandari P. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Piyush Rhajbhandari
Patan Hospital, Patan Academy of Health Sciences, Lalitpur, Nepal The research article is well written, however: Methodology: expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

Summary:
This retrospective study highlights an increasingly growing problem: antimicrobial resistance to antibiotics commonly used in hospitals, focusing on urinary infections. The objectives of this study were to identify the most frequent uropathogens, with their pattern of sensitivity to antibiotics, in patients with bacteriuria visiting the Beni hospital from April 2018 to April 2020. The results report an incidence of positivity equal to 14%. Escherichia coli (E. coli) was the most common isolate (74%), poly sensitive strain in 95% of cases, with high sensitivity towards Amikacin, gentamicin, ciprofloxacin but with a sensitivity of around 50% for the other antibiotics tested (ceftriaxone, cefixime, co-trimoxazole), and less than 40% to cefotaxime and ampicillin. In conclusion, E. coli is recognized as the most common pathogen associated with urinary tract infection, and indicating nitrofurantoin and amikacin as efficient antibiotics to be used in empirical therapy for the treatment of urinary tract infections in patients arriving at Beni hospital. The objectives of the study are very interesting, but the statistics and numbers of analyzed microorganisms do not fit the possibility of having a statistical analysis in terms of species isolated and antibiotic resistance profiles. Moreover, the manuscript needs a deeper reorganization and correction of the text.

Abstract:
The abstract is missing some information. This is part of the low number of microorganisms isolated. Taking apart the E. coli, all the other species are so limited that no assumption and evaluation can be included.

Introduction:
The introduction should be improved with epidemiological data of similar hospitals in terms of geographical region and assisted person.

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The aims and perspectives of the study should be described better.

Methods:
Concerning the high potential interest of people around the world to this kind of data, an international guideline in the antimicrobial resistance value should be used. Indeed in the paper, it was not possible to understand the concentration of the antibiotics that have been used. I strongly recommend the use of EUCAST lines.