Uropathogens Antimicrobial Sensitivity and Resistance Pattern From Outpatients in Balochistan, Pakistan

Objective To determine the pattern of microbes responsible for urinary tract infections and their susceptibility to different antibiotics. Method This is a cross-sectional study conducted at Quetta, Pakistan. The urine samples of 400 patients were collected and sent for culture and sensitivity analysis. The results were recorded on an excel datasheet. Descriptive statistics were used to describe the data. Results Out of 400 urine samples, 266 samples were culture positive for microorganisms. The most common organism on analysis was Escherichia coli 123/266 (46.24%) followed by Staphylococcus saprophyticus 59/266 (22.18%) and Klebsiella pneumonia 49/266 (18.42%). Gram-negative microorganisms were most susceptible to fosfomycin, cefoperazone/sulbactam, and meropenem. Gram-positive microorganisms were most susceptible to fosfomycin, cefoperazone/sulbactam, meropenem, and amoxicillin/clavulanate. High rates of resistance in E. coli were observed to most commonly prescribed broad-spectrum antibiotics; ceftriaxone (64.35%), cefotaxime (76.54%), ceftazidime (49.43%), cefepime (53.44%), levofloxacin (71.26%), and amoxicillin/clavulanate (70.31%). E. coli was the major multidrug-resistant organism. Conclusion High rates of antibiotic resistance and multi-drug resistance were revealed in this study due to the widespread and injudicious use of broad-spectrum antibiotics. Thus, it is highly recommended to regulate the pharmacies. Physicians should judiciously prescribe antibiotics and practice the culture and sensitivity of urine samples rather than blind prescription. Continued surveillance on uropathogens prevalence and resistance, new and next-generation antibiotics, and rapid diagnostic tests to differentiate viral from bacterial infections is the need of time.


Introduction
Urinary tract infections (UTIs) are among the most common types of infectious disease, accounting for approximately 150-250 million cases globally per year [1]. They are usually caused by gram-negative enteric rods, such as Escherichia coli (E. coli), Klebsiella, Proteus, etc. [2]. Approximately 50% of women acquire a UTI at least once in their lifetime [2]. The incidence of UTI among children is reported to be 30% all over the world [3]. 27% of women have a confirmed recurrence within the next six months, after the first episode of UTI [4]. UTIs can lead to renal scarring, ultimately leading to end-stage renal disease, therefore early diagnosis and treatment of UTI are necessary [5].
Antimicrobial resistance (AMR) is widespread across the globe, from the Americas to Australasia [6][7][8][9][10][11][12][13][14][15][16]. According to estimates by the Center for Disease Control (CDC), antibiotic-resistant bacteria cause at least 2.8 million illnesses and 35,000 deaths in the United States alone annually [17]. A study of trends of AMR in Europe observed that gram-negative uropathogens had high resistance to some of the most common antimicrobials. A north-to-south gradient in AMR exists in Europe, with higher resistance among southern European states like Greece, Cyprus, France, and Italy [18]. In a study of AMR in Asia pacific, "reduced sensitivity to commonly prescribed advanced-generation cephalosporin, piperacillin-tazobactam, and levofloxacin, among the studied gram-negative pathogens -Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp (ESKAPE) were observed" [16]. One of the causes of antibiotic resistance is the empirical treatment of UTI [9][10]. In India, resistant bacterial infections resulted in 58,000 infants deaths in 2013 [19]. In the first and second years of life, infants and toddlers spend around 40 days on antibiotics, which clearly illustrates antibiotic overuse [20]. 85% of children with acute otitis media, below two years of age, are prescribed antibiotics [21]. Around 500 million courses of antibiotics are used to treat both bacterial and viral diarrhea each year across four middle-income countries India, Indonesia, Nigeria, and Brazil [10]. Nevertheless, antibiotic abuse for a diarrheal disease is a leading cause of increasing antibiotic resistance (ABR) [22]. Therefore, the regional antibiotic susceptibility patterns of the microorganisms should be used for treatment. World Health Organization's global plan of action on AMR has stressed AMR surveillance across nations as an important strategy for countering AMR [23]. This study is one of the few addressing antibiotic susceptibility and resistance patterns in Baluchistan, Pakistan. More extensive AMR surveillance data is the need of time.

Materials And Methods
This is a cross-sectional study conducted at Quetta, Pakistan over a period of nine months. The sampling technique was non-probability consecutive sampling. Informed consent was taken from all the participants. The study was conducted in the outpatient department of the hospital. The inclusion criterion was patients presenting with symptoms of uncomplicated urinary tract infection. The exclusion criteria of the study were patients who had refused to participate, immunocompromised, patients suffering from phimosis or paraphimosis, uncircumcised males, and patients who had taken antibiotics within the past 24 hours. The urine samples of the patients were then taken to confirm the diagnosis. Early morning mid-stream samples of urine were collected in sterile containers and immediately processed for further procedures. Urine samples with significant bacterial growth ≥ 10 5 CFU/mL were considered positive. Colony study and biochemical tests were done to identify the microorganisms. MacConkey agar (Oxoid, England) was used to subculture the colonies to get pure growth of the microorganisms. Biochemical tests were done to identify the microorganisms. Tests performed for Gram-positive cocci include catalase, coagulase, and novobiocin test. Tests performed for gram-negative bacilli include Simmon's citrate agar, methyl red, urease, sulfur indole motility test, and triple sugar iron test. Kirby-Bauer disk diffusion method was used to determine the antibiotic susceptibility of the isolated colonies. Müller-Hinton agar plates were used to identify the sensitivity pattern. After this, the measurement of the zone of inhibition of bacterial growth was performed and a comparison was done with the guidelines of the Clinical and Laboratory Standards Institute (CLSI, 2018).
The organisms were subjected to various groups of antibiotics including penicillins, fluoroquinolones, aminoglycosides, cephalosporins, and tetracyclines. In addition, based on previous urine culture studies in Pakistan, due to high rates of resistance to recommended antibiotics, fosfomycin, meropenem, and vancomycin were also tested. The culture and sensitivity results for the specimens were recorded on a google form and an excel sheet was produced. The excel data sheet was analyzed for sensitivity and resistance patterns of microorganisms. Descriptive statistics were used to describe the data in terms of numbers and percentages

Results
Out of 400 urine samples, 266 samples were culture positive for microorganisms. 85 culture-positive samples were obtained from male participants and 181 culture-positive samples from female participants. E. coli was the most frequently observed microorganism followed by Staphylococcus saprophyticus and Klebsiella pneumoniae. The details of the frequency of uropathogens in urinary samples are shown in Table 1.   Gram-negative bacteria were more common than gram-positive in males (78.82%) and females (64.64%). Table 3 depicts the microorganism's gram stain distribution pattern based on gender.   The multidrug-resistant (MDR) pattern was also explored. Some of the samples were resistant to more than one antibiotic of the same or different groups. E. coli was the major multidrug-resistant organism, MDR in E. coli ranged from 1.6-17% while in Staphylococcus saprophyticus it ranged from 1.7-18.64%. Thirty-eight (14.28%) out of the 266 samples were multidrug-resistant to cefotaxime, ceftazidime, and ceftriaxone. 35 (13.15%) samples were multidrug-resistant to cefotaxime, ceftazidime, and ciprofloxacin. Twenty-six (9.77%) samples were multidrug-resistant to amoxicillin/clavulanate, ceftriaxone, and levofloxacin. Seventeen (6.3%) samples were multidrug-resistant to gentamycin, ciprofloxacin, and ceftriaxone. 15 (5.6%) samples were resistant to amoxicillin/clavulanate, ceftriaxone, and doxycycline. Eight (3%) samples were multidrug-resistant to four antibiotics, amoxicillin/clavulanate, levofloxacin, ceftriaxone, and doxycycline. Four (1.5%) of urine samples were resistant to amoxicillin/clavulanate, gentamycin, ciprofloxacin, ceftriaxone. Three (1.1%) urine samples were resistant to gentamycin, ceftriaxone, ciprofloxacin, and fosfomycin.

Discussion
UTIs are caused by several microorganisms. E. coli (46.2%) followed by coagulase-negative staphylococci (CONS) (30.6%) and Klebsiella pneumoniae (18.4 %) were the most common uropathogens in our study. A brief review of the most common uropathogens isolated from urine culture studies in Pakistan is as follows.
Alarming rates of antibiotic resistance have been observed by other researchers in Pakistan. A brief description of such studies is reviewed here. Farooqi et al. observed that in the seven years study period from 1990-1997, the microorganisms were observed to have a low rate of resistance to cefotaxime, ceftriaxone, aztreonam, ofloxacin, amikacin, gentamicin, and nitrofurantoin, despite an increase in antibiotic resistance [24]. However, antibiotic resistance has increased now to the most commonly prescribed antibiotics as observed by more recent studies. According to a study by Muzamil et al., "almost more than 60% of the total sample were resistant to co-amoxiclav, cefotaxime, and ceftriaxone, piperacillin/tazobactam, cefoperazone/sulbactam, and ciprofloxacin. All 100% cultures were resistant to amoxicillin" [25]. Their findings are similar to our study except for cefoperazone/sulbactam which showed better sensitivity in our study. Malik et al. concluded that "the rates of resistance of amoxicillin/clavulanate, ciprofloxacin, ceftriaxone, ceftazidime were almost 70 %. The cultures were more than 90% sensitive to amikacin, fosfomycin, and nitrofurantoin. Imipenem showed a sensitivity of 85%" [26]. The rates of resistance and sensitivities in their study are similar to ours except for nitrofurantoin which was not tested in our study . In their study Rizvi et al. observed that "more than 75% of the strains were resistant to fluoroquinolones. 63% of E. coli samples were found resistant to ceftriaxone and cefepime. However, the resistance profile towards nitrofurantoin, fosfomycin, and carbapenem was less than 10.7%." [27]. In their study, Inam Ullah Khan et al. reported "The susceptibility pattern of E. coli showed that approximately more than 80% of the bacterial isolates were sensitive to imipenem, amikacin, tazobactam/piperacillin, and 69% to nitrofurantoin. Sensitivity to commonly used oral antibiotics was very low" [28]. Basharat Ali Khan et al. observed that "Out of 246 patients (20.43%) with nosocomial urinary tract infections, almost more than 60% of the samples were resistant to cefotaxime, ciprofloxacin, and gentamycin [29].
All the aforementioned studies indicate rising antibiotic resistance to some of the most commonly prescribed and broad-spectrum antibiotics. The high level of resistance is alarming when compared to developed countries like America. The prevalence of extended-spectrum beta-lactamase producers among the Enterobacteriaceae was less than 10% in North America, much lower as compared to Asia and the Middle East countries (>40%), according to "Study for Monitoring Antimicrobial Resistance Trends (SMART)" [30]. Health authorities, physicians, and pharmacies should work together to tackle this problem, regulate the healthcare industry especially prescription and purchase of antibiotics. Anyone in Pakistan can selfprescribe antibiotics and pharmacies dispense them without any prescription. Self-medication's overall prevalence was reported to be 39% in a review of 34 studies from low-middle-income countries with 31,340 participants, [31]. The short duration of treatment, insufficient doses, taking the wrong medicine and inappropriate sharing of medicines were some of the most common malpractices [31]. On the other hand, doctors also overprescribe antibiotics, without following standard treatment algorithms. America's national strategy to combat antibiotic resistance is an exemplary initiative for other countries. It recommends that "Improved detection can be achieved through appropriate data sharing, expansion, and coordination of existing surveillance systems, and establishment of a standardized platform for resistance testing and genetic characterization of bacteria. Development of Rapid "point-of-need" tests to distinguish between viral and bacterial infections and identifying bacterial drug sensitivities, new and next-generation antibiotics, diagnostics, and vaccines is the need of time. Rapid detection and control of outbreaks is another strategy to combat AMR. Global collaboration and capacities should be enhanced" [32].
Despite all efforts, certain limitations exist in the study. First, the urine culture and sensitivity data are from a single institution with smaller sample size, and can not be generalized to all UTI patients. Secondly, the data is primarily focused on urine culture findings, therefore, epidemiologic and demographic characteristics and comorbid conditions of patients are not extensively described.