Antimicrobial Susceptibility Pattern of Uropathogens Isolated at a Private Hospital in Khulna, Bangladesh

Background & objectives: Urinary tract infection (UTI) is one of the most common infectious diseases affecting all age & sex groups, causing significant morbidity & mortality with a substantial economic burden. Due to the irrational empiric use of antibiotics, resistance is increasing at an alarming rate. Bacteria causing UTIs & their antimicrobial susceptibility pattern varies among different areas & changes frequently over time. This study aimed to identify microorganisms causing UTI at a private hospital in the southern part of Bangladesh & to determine their sensitivity pattern towards commonly used antibiotics. Methods: A cross-sectional study was conducted in the clinical pathology & microbiology laboratory at Islami Bank Hospital (IBH), Khulna during the period of January’2021 to July’2021. Clean catch midstream urine samples (MSU) from 221 clinically suspected UTI cases of all age & sex groups were included in the study. Uropathogens were isolated & identified by standard microbiological techniques & susceptibility was determined by Kirby Kirby-Bauer disc diffusion method. Results: Among 221 suspected UTI cases, 108 (48.8%) were positive for urine culture, of which 103 (95.37%) showed antimicrobial growth. The majority 75 (69.4%) of the culture-positive cases were female. E. coli 50(44.24%) was the most predominant bacterial isolate, followed by Klebsiella 18(15.92%) & Enterococcus 16(14.15%). All Isolated gram-positive cocci (GPC) showed very high sensitivity to Vancomycin (83.3% to 100%), Linezolid (87.5% to 100%) & moderate sensitivity to Meropenem (66.7% to 75%). However, they showed relatively lower sensitivity to Ciprofloxacin, Doxycycline, Gentamicin, and Amoxiclav & very poor sensitivity to Nitrofurantoin (31.3% to 50% only). Levofloxacin had relatively better sensitivity against GPC (S. aureus-83.3% & CONS-75%), except Enterococcus. All gram-negative bacilli (GNB) showed a very good sensitivity towards Amikacin & Meropenem, ranging from 77.8% to 100%.


Introduction
Urinary tract infection (UTI) remains one of the most frequently encountered infectious diseases in medical practice, occurring from neonatal to geriatric age group. 1 It acts as a major cause of morbidity in both community & hospital settings affecting around 405 million people globally 2 with nearly 150 million diagnosed cases each year, costing the global economy over 6 billion US dollars. 3UTI also accounts for more than 40% of the total cases of nosocomial infections reported by acute care hospitals & 66% to 86% of these infections are caused as an aftereffect of instrumentation of the urinary tract, mainly catheterization. 4cteria remain the major causative organisms of UTIs and are accountable for more than 95% of the cases. 5Growth of more than 105 colony-forming unit (CFU) of bacteria per ml of urine for asymptomatic individuals & 103 for symptomatic individuals refers to significant bacteriuria. 6Gram-negative bacilli are mostly accountable for UTI 7 , of them E. coli is the predominant organism, which is responsible for more than 75% of the reported cases in both outpatient & inpatient facilities. 8Other important Gram-negative organisms causing UTI are Klebsiella, Enterobacter, Citrobacter, Proteus, Serratia as well as Pseudomonas species.Among gram-positive organisms the most frequently isolated pathogens are Coagulase negative staphylococcus (CONS), S. aureus & Enterococcus species. 9owadays, multidrug-resistant E. coli & K. pneumoniae are frequently recognized to be responsible for nosocomial infections including catheterassociated UTI (CA-UTI). 10Most of the UTI cases are usually antimicrobial (caused by a single bacterial species) but polymicrobial infections (mixed bacterial infections) are also reported. 11e incidence of UTI is more common in women than men & it is reported that about 35% of healthy women suffer from UTI at some point in their lifetime.Most UTI cases are caused by the retrograde ascent of fecal bacterial flora to the urinary bladder & kidney via the urethra, notably in females as their urethra is shorter & wider than men's.UTIs in female are also very common due to their vaginal colonization with uropathogens 7 .Susceptibility to trauma during coitus, pregnancy & obstruction may also contribute to the higher incidence of UTIs in females. 11olation & identification of uropathogens followed by their antibiotic susceptibility pattern is obtained by doing urine culture.As a common practice, empirical antibiotic therapy is initiated before the urine culture report is available, which leads to the indiscriminate use of antibiotics, resulting in the emergence of resistant microorganisms to one or more of the available drugs.As a result, there is a gradual narrowing of the scope for effective antibiotics to combat bacterial infections like UTIs. 7In addition, the pattern of antimicrobial susceptibility of uropathogens is regularly changing due to ever-increasing uses of antibiotics for the treatment of different variety of infections outside the urinary tract. 1,11Bacteria producing extended-spectrum beta-lactamases (ESBLs) are constantly increasing in the population, which shows resistance to most of the broad-spectrum antibiotics except carbapenem. 12 alarming picture has been reflected by a recent study in Bangladesh, which demonstrated more than 75% of the E. coli causing UTIs are resistant to third-generation cephalosporins. 13In Bangladesh UTI is a significant public health problem & increasing antibiotic resistance even complicates the treatment of uncomplicated UTI by increasing patient morbidity & health care costs due to frequent treatment failure, recurrent infections & unnecessary use of broad-spectrum antimicrobials. 10,14There is no large-scale prospective survey of UTIs in Bangladesh, that can reflect the up-to-date burden of the infections & the antimicrobial susceptibility pattern in the community. 10Nationwide continuous monitoring of the etiology of the infections & susceptibility pattern is of paramount importance for not only selecting appropriate antibiotics but also for rational choice of empiric therapy to reduce the misuse or overuse of antibiotics. 11his study was carried out to determine the recent bacterial etiology for UTIs & to analyze their susceptibility pattern in an acute care hospital in Khulna City to disseminate information about the choice of appropriate antibiotics for empirical therapy, which will guide our physicians in treating UTIs.Clean catch midstream urine samples (MSU) (4-5 ml) were collected in a sterile disposable leakproof container from all the enrolled suspected UTI cases and transported immediately to the laboratory.Urine culture was done by semi-quantitative method on MacConkey agar, Blood agar, and Chromogenic media by using calibrated loops and incubated aerobically for 24 hours at 37°C. 1,7A routine microscopic examination of all urine samples was done to count pus cells.If no colony appeared after 24 hours of incubation, those culture plates were further incubated for 48 hours.Bacterial isolates were identified and confirmed by using standard microbiological and biochemical tests like Gram staining, examining colony morphology on culture media, motility indole urease test, citrate utilization test, by observing biochemical changes in TSI media, catalase, coagulase & oxidase test. 7timicrobial susceptibility testing was performed on Mueller Hinton agar using disk diffusion Kirby Bauer's technique according to Clinical and Laboratory Standards Institute (CLSI) guidelines.Antibiotics were interpreted as sensitive & resistant, based on the zone of inhibition of bacterial growth, recommended by the disc manufacturer.Gentamicin (10µg), Ciprofloxacin (5µg), Levofloxacin (5µg), Amoxiclav (30µg), Ceftriaxone (30µg), Meropenem (30µg) & Nitrofurantoin (300µg)-these 7 antibiotic discs were used for both gram-positive & gram-negative organisms.Doxycycline (30µg), Cefepime (30µg), Vancomycin (30µg) & Linezolid (30µg) discs were used only for gram-positive bacteria.On the other hand, Amikacin (30 µg), Azithromycin (15µg), Cefotaxime (30 µg), Cefuroxime (30 µg), Ceftazidime (30µg) & Piperacillin-tazobactam (110 µg) discs were used only for gram-negative organisms.

Discussion
The present study demonstrated a higher percentage of female patients (62.44%) over male patients (37.56%) among the total suspected UTI cases.This finding corroborates well with a recent report from a teaching hospital in Bangladesh, where among the suspected UTI cases majority (59.37%) were female & 40.63% were male. 11 documented 48.87% (108/221) cases were positive for urine culture, which is almost similar to the study done at a tertiary care hospital in Uttarakhand, India having 49.1% (166/338) positivity for urine culture. 15Sharmin et al. documented 46.66% positive urine culture also among children, which is very much consistent with present findings. 16In contrast, a slightly higher (55%) urine culture positivity was reported by Biswas et al. 17 & a relatively lower rate (36.1%) of positive urine culture was recorded by a study at a tertiary care hospital in Haripur, Pakistan.The inclusion of only outpatients' samples may explain this lower rate of positive urine culture in this study. 18On the contrary to our study, a report from a tertiary care hospital in Dhaka city reflected a very high rate (90.37%) of positive urine culture.The inclusion of urine samples having significant pyuria (≥ 5 pus cells /HPF) from catheterized UTI patients can be a good explanation for this very high rate of positive culture. 19is study recorded that the majority (69.44%) of culture-positive patients were female.A study at a tertiary care hospital in Dhaka, Bangladesh also showed female predominance (58.2%) among culture-positive UTI patients. 1Ascending infection due to short urethra, the wrong practice of cleaning the perineum forward from the anus to vulva & sexual intercourse may be responsible for this higher frequency of UTI in females. 7In contrast to our findings, Arina et al. reported that all GNB were highly resistant to Gentamicin (E.coli-57.8%,Klebsiella-57.1%,Pseudomonas-56.41%& Proteus-61.1 resistant), except Enterobacter which was only 31.2% resistant to Gentamicin. 19 found a lower sensitivity pattern of GNB towards Amoxiclav (E.coli-56%, klebsiella -38.8 %, pseudomonas -11.1 %, Enterobacter-25% & proteus-0.0%).GNB had a very similar lower sensitivity trend to Cefuroxime also.GNB were also poorly sensitive to Azithromycin (E.coli-34%, Klebsiella & proteus-50%, Pseudomonas-33.3%,Enterobacter-25%).This is in agreement the with study of Khanam et al., who also reported a much lower sensitivity pattern of GNB towards Amoxiclav, azithromycin & cefuroxime. 1 Isolated GNB showed poor sensitivity to Ciprofloxacin (Klebsiella & pseudomonas-44.4%,Enterobacter & Proteus-50%), except E. coli which showed slightly higher sensitivity (64%), which is very much consistent with the findings of Sohely et al. 16 E. coli, Klebsiella & proteus showed relatively higher sensitivity to Levofloxacin (70%, 61.1% & 100% respectively) but sensitivity pattern of Enterobacter & Pseudomonas towards Levofloxacin was as low as their sensitivity to ciprofloxacin.This is also true for the study done by Ritu Saha et al. 7 & Nahar et al. 24 The present study showed that Klebsiella & Proteus had lower sensitivity to nitrofurantoin (  26 , which is again in agreement with our study. We found that another 3rd generation of cephalosporin-ceftazidime displayed a very low sensitivity against Klebsiella (33.3%),Enterobacter (25%), Proteus (0.0%) & relatively higher sensitivity against E. coli (58%) & Pseudomonas (66.6%).Akram et al. reported that ceftazidime showed good sensitivity against E. coli (69%) & Pseudomonas (67%), which is almost similar to our documentation. 25On the contrary, Khanam et al. reported relatively lower sensitivity of E. coli (50%) & Pseudomonas (30%) to ceftazidime. 1 A study done on catheterized patients in a tertiary care hospital in Bangladesh, documented lower sensitivity of Klebsiella (33.34%), and Enterobacter (25%) to ceftazidime, which is completely consistent with our report.That study also showed lower sensitivity of E. coli (22.1%) & Pseudomonas (28.2%) to ceftazidime 19 , which is much opposite to our findings.
Gradually, uropathogens are acquiring resistance to the most frequently used antimicrobials 16 , which is reflected in our as well as in other studies.Selective drug pressure is one of the most important causes behind the occurrence of resistant bacterial strains.The use of antibiotics (like-cotrimoxazole, and ciprofloxacin) in livestock is also responsible for the emergence of antimicrobial resistance. 27In Bangladesh, dispensing of antibiotics is not restricted to prescriptions of registered physicians only, rather they are widely available over the counter throughout the country.It leads to the irrational use of many life-saving antibiotics, triggering the emergence of drug-resistant mutants. 28Actually, antibiotic Sensitivity patterns are altering day by day, which varies from one healthcare center to another even within the same city 16 & also significantly varies between different geographic areas. 29So, proper knowledge of the antimicrobial sensitivity pattern of uropathogens in a particular area is of paramount importance in designing effective antibiotic therapy. 7is study was done by analyzing the retrospective laboratory data only.So, one of the important shortcomings of our study was that we failed to categorize UTI patients whether complicated or uncomplicated, due to lack of clinical information.Moreover, we could not separate community-acquired, nosocomial & catheter-associated UTIs.We admit that some bacterial species could not be isolated & identified due to lack of laboratory facilities.Furthermore, MIC was not done for the determination of antimicrobial susceptibility.

Conclusion & Recommendations
E. coli, Klebsiella & Enterococcus spp.are the most frequently isolated uropathogens at our hospital.UTI with CONS cannot be overlooked also.
Amikacin, meropenem, piperacillintazobactam & sometimes gentamicin can be a good choice for UTIs caused by gram-negative bacilli.Levofloxacin can also be considered except for Pseudomonas spp.For gram-positive cocci vancomycin, linezolid are most sensitive molecule, but it must be used with caution to avoid the emergence of resistance.3rd generation cephalosporins failed to show promising responses to both gram-positive & negative organisms.Nitrofurantoin was not so effective against gram-positive organisms but it showed a good response to some gram-negative bacilli.
As uropathogens are gaining resistance to commonly used antimicrobials in developed as well as developing countries, sensitivity patterns in a defined geographical area should be determined annually to establish an effective guideline for physicians.For treating UTIs, antibiotics must be selected according to the culture & sensitivity report.Physicians should prescribe empirical therapy, considering the recent antibiogram of a particular area.Moreover, it is also very essential to develop a national policy to undertake 'Antimicrobial stewardship programs' including all levels of healthcare providers to prevent the occurrence of drug resistance.
was a retrospective cross-sectional study conducted in the clinical pathology & microbiology laboratory at Islami Bank Hospital (IBH), Khulna during the period of January'2021 to July'2021.Clinically suspected 221 UTI cases irrespective of age & sex, referred from outpatient & different inpatient departments of the hospital with the requisition of urine culture & sensitivity were included in the study.