Prevalence and Antibiotic Susceptibility Profiling of MDR P. aeruginosa Vol. 13 (1), June 2022 ISSN (Print): 2305 – 8722 ISSN (Online): 2521 – 8573 R A D S J . B i o l . R e s . A p p l . S c i . 1 Op e n Ac c e s s F u l l L e n g t h A r t i c l e Prevalence and Antibiotic Susceptibility Profiling of MDR Pseudomonas aeruginosa from UTI Patients of Southern Punjab, Pakistan Iqra Arooj1, ⃰, Aysha Asghar1, Mehvish Javed1, Amina Elahi2, Asghar Javaid3 1Department of Microbiology & Molecular Genetics, The Women University, Multan, Pakistan. 2University Institute of Medical Laboratory Technology, University of Lahore, Lahore, Pakistan. 3Department of Pathology, Nishtar Medical University, Multan, Pakistan. A B S T R A C T Background: Urinary Tract Infections (UTIs) are of major concern in health care settings. An increasing percentage of organisms causing nosocomial infections have now become multidrug resistant. Hence, constant surveillance of etiology and antimicrobial susceptibility of uropathogens including Pseudomonas aeruginosa is compulsory. Objective: Main purpose of this study was to assess the prevalence rate of various pathogens, and to evaluate the multidrug resistance pattern of Pseudomonas aeruginosa in UTI patients of Southern Punjab, Pakistan. Methodology: A cross-sectional study was performed to obtain quantitative data of UTI patients, for which one hundred and fifty (150) urine samples were obtained from UTI patients admitted to Nishtar Hospital, Multan. Samples were processed for culture, bacterial isolation and identification. Antibiotic sensitivity of P. aeruginosa isolates was determined in agreement with Clinical Laboratory Standard Institute Guidelines. Results: Prevalence of UTI was greater in females (59%) when compared to males (41%). Among 150 urine samples, uropathogens isolated were Escherichia coli-50(33.33%), followed by Klebsiella pneumoniae-27(18%), Proteus spp.-15(10), Staphylococcus saprophyticus-13(8.66%), Staphylococcus aureus-11(7.33%), Pseudomonas aeruginosa-10(6.66%), Enterobacter spp.-9(6%), Candida albicans-8(5.33%) and Citrobacter spp.-7(4.66%). Antibiotic sensitivity profiling of Pseudomonas aeruginosa revealed highest sensitivity to cefixime (80%), followed by gentamicin (70%), piperacillin/tazobactam (60%) and nitrofurantoin (40%). Conversely, maximum resistance was observed against amikacin (80%), followed by ciprofloxacin (60%), imipenem (50%) and amoxicillin (50%). Conclusion: Knowledge of dynamic etiology and ever-changing drug resistance patterns is essential for appropriate management and treatment of UTIs necessitating their exploration on a regular basis. Keywords Antibiotic sensitivity, Multidrug resistance, Nosocomial infections, Pseudomonas aeruginosa, Susceptibility, Urinary tract infection. *Address of Correspondence iqra.6051@wum.edu.pk Article info. Received: April 04, 2021 Accepted: February 04, 2022 Cite this article Arooj I, Asghar A, Javed M, Elahi A, Javaid A. Prevalence and Antibiotic Susceptibility Profiling of MDR Pseudomonas aeruginosa from UTI Patients of Southern Punjab, Pakistan. 2022; 13(1):1-9. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium provided the original work is properly cited. O R I G I N A L A R T I C L E Prevalence and Antibiotic Susceptibility Profiling of MDR P. aeruginosa Vol. 13 (1), June 2022 ISSN (Print): 2305 – 8722 ISSN (Online): 2521 – 8573 R A D S J . B i o l . R e s . A p p l . S c i . 2 I N T R O D U C T I O N Urinary Tract Infections (UTIs) have been reported as one of the most predominant microbial infections globally, that can be diagnosed both in the outdoor and in the clinics1. These infections are generally categorized into hospital and community-acquired infections2. Patients develop a high risk of acquiring hospital-borne UTIs when admitted to hospital for 48hrs or longer3. Calculated percentage of UTIs that are acquired from hospitals is 35% in admitted patients4. In UTI patients, microorganisms can affect any part of urinary system by weakening natural body defense mechanisms which can be diagnosed depending on the infection site as pyelonephritis (inflammation of kidneys), cystitis (inflammation of bladder), and urethritis (inflammation of urethra), respectively5. On average, 95% of UTI cases are reported because of the presence of single microbial species, while more than one specie is usually present in urine culture due to contamination6. Out of all, 80-85% of the UTI cases are attributed towards gram negative bacteria including Escherichia coli, Klebsiella pneumoniae, Enterobacter spp. and Pseudomonas aeruginosa, however the most frequently encountered pathogen among these is E. coli7. Specifically, among catheterized patients, the major causative agent is P. aeruginosa, a gram-negative pathogen considered as superbug in numerous human infections8. Its opportunistic behavior and ubiquitous existence enable it to become associated with pathogenesis particularly in immunocompromised patients9. P. aeruginosa is held responsible for 11% of nosocomial infections and it produces a multitude of virulence factors that enable its attachment to host cell surface, facilitating entry into the host tissues and suppression of host immunity10. In addition to virulence factors, P. aeruginosa produces biofilms and chemical signals for quorum sensing on the surface of urinary catheters leading towards the development of infections11. Antimicrobial resistance is one of the most significant and challenging issues emerging with regard to hospital as well as community-acquired UTIs12. Continuously rising drug resistance rate is creating complications with reference to treatment, morbidity rate, duration of stay in hospital, etc.13. Rate of UTIs varies between developing and developed countries depending on random usage of antibiotics. Antibiotic resistance against common bacteria has become a huge trial for clinicians in our part of world in terms of devising therapeutic strategies against UTIs14. The leading cause of increasing resistance rate is mutation and resistance gene transfer among pathogens. Plasmids and transposons are the carriers of resistance genes making single type of organism resistant to several drugs precipitating in the emergence of Multiple Drug Resistant (MDR) pathogens15. Recently, continuous increase in antibiotic resistance has been observed particularly against ampicillin and trimethoprim which is adversely affecting the management and treatment of UTIs16. Furthermore, encountering pathogenic infections that are contagious on routine basis is aggravating the problems while pathogens are becoming resistant to first-line antimicrobial agents17. It is hereby critical for physicians to recommend appropriate treatment in accordance with the susceptibility pattern of causative agents of UTIs18. When infection is in a specific area, an organized empirical therapy plan is required to reduce spread of resistance among pathogens, for which knowledge of prevalence rate of particular pathogens and their most recent antimicrobial susceptibility profile is compulsory19. Keeping this in view, we aimed to isolate, purify, and identify P. aeruginosa which is the predominant cause of UTIs among catheterized patients. Furthermore, we evaluated the antibiotic susceptibility profile of P. aeruginosa isolates in order to estimate emerging sensitivity pattern in the local population of Southern Punjab, Pakistan where 10% of population is speculated to suffer from UTI at some stage of their life20. M A T E R I A L S & M E T H O D S Sample Collection One hundred and fifty (150) samples of midstream urine were collected in sterilized containers from the UTI patients bedded in Nishtar Hospital, Multan for this hospital-based study after gaining ethical approval from the Ethical Committee of Nishtar Hospital. The main inclusion criterion was referral and admission to nephrology, urology, gynecology, neurology, & medicine wards of Nishtar Hospital in addition to catheterization and non- catheterization as well as both genders. Further, inclusion Prevalence and Antibiotic Susceptibility Profiling of MDR P. aeruginosa Vol. 13 (1), June 2022 ISSN (Print): 2305 – 8722 ISSN (Online): 2521 – 8573 R A D S J . B i o l . R e s . A p p l . S c i . 3 was based on appearance of symptoms within two days of stay in the hospital and diagnosis by physicians. All those cases showing symptoms of infection prior to 48hrs stay in the hospital, and/or diagnosed with infections other than UTI were excluded from the study. Personal information including demography and geography as well as medical information encompassing symptoms of patients was documented on a pre-designed questionnaire proforma signed by patients to ascertain informed consent. The samples were processed in microbiology section, pathology department of Nishtar Medical University, Multan, under standardized procedures to reduce the chances of contamination. Isolation and Identification of P. aeruginosa For the purpose of microscopic examination, centrifugation of urine samples was performed at 5000rpm for 5mins and sediment was considered for standard operating procedures. Cases with considerable pus cells (≥10 WBCs/mm) were administered for further analysis. For isolating and identifying uropathogens, urine samples were streaked onto Cysteine Lactose Electrolyte Deficient (CLED) agar plates maintaining sterility and incubated at 37ºC for 18-24hrs under ventilated conditions as CLED media is strongly recommended for isolation, identification, and quantification of the uropathogens. Subsequent to incubation, colony characteristics were observed by using hand lens. P. aeruginosa colonies on CLED medium were light green with typical intertwined surface, while colonies of E. coli were yellow due to its ability to ferment lactose. Colonies of Klebsiella spp. were also yellow and enormously mucoid in terms of appearance. Colonies of Proteus spp. were radiant blue and those of S. aureus were deep yellow. Gram staining was done subsequently to observe gram-negative rods of P. aeruginosa. Various biochemical tests comprising of triple sugar iron, oxidase, motility, citrate utilization, methyl red / Voges-Proskauer and indole tests were performed for species identification. Antimicrobial Susceptibility Profiling Antimicrobial susceptibility pattern of P. aeruginosa isolates was determined by performing Kirby-Bauer method (disc diffusion assay) using Mueller Hinton (MH) agar. About 3-5 colonies from pure cultures of P. aeruginosa isolates were transferred to a sterilized test tube containing 5ml normal saline, with the help of sterile wire loop and this suspension of colonies was compared with 0.5 McFarland standard. With the help of sterile cotton swab dipped in microbial suspension, each MH agar plate was inoculated and antibiotic discs were applied to it at specific and appropriate distance. The plates were then incubated at 37ºC for 24hrs subsequent to which antibiotic sensitivity zones were measured in mm by ruler and recorded as sensitive, intermediate sensitive or resistant following the Clinical and Laboratory Standard Institute (CLSI) guidelines. Isolates were evaluated for their susceptibility or resistance towards amikacin (AK), ciprofloxacin (CIP), imipenem (IPM), amoxicillin (AMX), cefixime (CFM), gentamicin (CN), piperacillin/tazobactam (TZP), and nitrofurantoin (NF). R E S U L T S Baseline Characteristics of Study Population Of the 150 urine samples of patients admitted to different wards of Nishtar hospital collected and investigated for the presence of uropathogens, 88 were females whereas 62 were males. Out of all the females, 58 were associated with catheter while 30 were not associated with catheter. Out of all the males, 35 were associated with catheter and 27 were not associated with catheter. Patients belonged to different regions of Multan and nearby localities (Fig. 1). This highlights the fact that patients from all over South Punjab become admitted to Nishtar Hospital, Multan, thereby making our sample population representative of entire Southern Punjab region. Symptomatic frequency was observed to be greater in females when compared to males as evidenced by the fact that frequency of urination, burning sensation during urination, flanking pain and dysuria were all more frequently experienced by females in comparison with males (Fig. 2). Information regarding prevalence of various risk factors associated with UTI was also documented and it was observed that indwelling catheter was the major risk factor associated with UTIs followed by pregnancy, presence of stone, enlarged prostate, diabetes, injured spinal cord and presence of tumor in kidney, respectively (Fig. 3). Prevalence and Antibiotic Susceptibility Profiling of MDR P. aeruginosa Vol. 13 (1), June 2022 ISSN (Print): 2305 – 8722 ISSN (Online): 2521 – 8573 R A D S J . B i o l . R e s . A p p l . S c i . 4 Figure 1. Geographical distribution of study population. Figure 2. Gender-based prevalence of symptoms associated with UTI in study population. Figure 3. Prevalence of risk factors associated with UTI among study participants. Prevalence and Antibiotic Susceptibility Profiling of MDR P. aeruginosa Vol. 13 (1), June 2022 ISSN (Print): 2305 – 8722 ISSN (Online): 2521 – 8573 R A D S J . B i o l . R e s . A p p l . S c i . 5 Age and Gender Based Prevalence of UTIs UTIs were recorded as being more prevalent among females (59%) as compared to males (41%) in the study population highlighting the substantial gender-based differences in prevalence of UTI. Furthermore, UTI frequency was observed as being higher among younger females and males as compared to older males and females, in general (Table 1). In both genders, UTIs were the most prevalent among individuals that were 20 to 30 years old, and its frequency constantly decreased while females grew older to 70 years and males grew older to 60 years. A slight increase in UTI frequency was observed among females older than 70 years and males older than 60 years. Prevalence of Microbes in UTI Patients Based on the urine cultures, it was confirmed that in all urine samples, at least one kind of urinary microbe was present. Isolates from various samples included P. aeruginosa, E. coli, Enterobacter spp., Klebsiella spp., Proteus spp., S. aureus, S. saprophyticus, Candida albicans and Citrobacter spp. It was observed that E. coli was the most predominant uropathogen among males as well as females followed by K. pneumoniae, Proteus spp., S. saprophyticus, S. aureus, P. aeruginosa, Enterobacter spp., C. albicans and Citrobacter spp., respectively (Table 2). Table 1. Age and Gender Based Prevalence of UTI in the Study Population. Age in Years Males (N=62) Females (N=88) 20-30, N (%) 29 (46.77) 44 (50) 30-40, N (%) 12 (19.35) 20 (22.72) 40-50, N (%) 8 (12.90) 11 (12.50) 50-60, N (%) 3 (4.83) 3 (3.40) 60-70, N (%) 4 (6.45) 3 (3.40) Above 70, N (%) 6 (9.67) 7 (7.95) N = Number of individuals Table 2. Gender Based Prevalence of Uropathogens Isolated from UTI Patients Included in the Study. Uropathogen Total (N=150) Females (N=88) Males (N=62) E. coli, N (%) 50 (33.33) 31 (35.22) 19 (30.64) K. pneumoniae, N (%) 27 (18) 17 (19.31) 10 (16.12) Proteus spp., N (%) 15 (10) 8 (9.09) 7 (11.29) S. saprophyticus, N (%) 13(8.66) 7 (7.95) 6 (9.67) S. aureus, N (%) 11 (7.33) 6 (6.81) 5 (8.06) P. aeruginosa, N (%) 10 (6.66) 6 (6.81) 4 (6.45) Enterobacter spp., N (%) 9 (6) 5 (5.68) 4 (6.45) Candida spp., N (%) 8 (5.33) 4 (4.54) 4 (6.45) Citrobacter spp., N (%) 7 (4.66) 4 (4.54) 3 (4.83) N = Number of individuals Prevalence and Antibiotic Susceptibility Profiling of MDR P. aeruginosa Vol. 13 (1), June 2022 ISSN (Print): 2305 – 8722 ISSN (Online): 2521 – 8573 R A D S J . B i o l . R e s . A p p l . S c i . 6 Table 3. Antibiotics used against Isolates of P. aeruginosa with Their Mechanism of Action and Prevalence in Study Participants. Antibiotic group Antibiotic Mechanism of action Code Prevalence ( ⃰ S or ⃰ R) Aminoglycoside Amikacin Inhibitor of protein synthesis AK 80% (*R) Fluoroquinolone Ciprofloxacin Inhibitor of nucleic acid synthesis CIP 60% (*R) Carbapenem (Beta- lectamase inhibitor) Imipenem Inhibitor of cell wall synthesis IPM 50% (*R) Beta-lactamase inhibitor Amoxicillin Inhibitor of cell wall synthesis AMX 50% (*R) Cephalosporin Cefixime Inhibitor of cell wall synthesis CFM 80% (*S) Aminoglycoside Gentamicin Inhibitor of protein synthesis CN 70% (*S) Beta-lactamase inhibitor Piperacillin/ Tazobactam Inhibitor of cell wall synthesis TZP 60% (*S) Urinary tract antiseptic Nitrofurantoin Inhibitor of cell wall and nucleic acid synthesis NF 40% (*S) *R=Resistant; S=Sensitive Antibiotic Susceptibility Profile of P. aeruginosa Antibiotic susceptibility pattern was determined specifically for P. aeruginosa isolates and the results have been summarized in Table 3. Cefixime, which is a cephalosporin antibiotic, was demonstrated to be the most effective of all the drugs tested against P. aeruginosa isolates whereas amikacin, an aminoglycoside, was recorded as being the least effective drug in this regard. In terms of effectiveness against P. aeruginosa uropathogen, cefixime was followed by gentamicin, piperacillin/tazobactam and nitrofurantoin, respectively. With reference to ineffectiveness, amikacin was followed by ciprofloxacin, imipenem and amoxicillin, respectively. Furthermore, majority (90%) of the isolates had become resistant to two or more antibiotics classifying them as MDR pathogens. D I S C U S S I O N P. aeruginosa was conferred for the first time in 1882 by Gessard when he isolated it from green pus8. Urinary tract is the most prevalent site of healthcare-associated infections, with P. aeruginosa accounting for 7-10% of UTIs diagnosed in patients admitted to hospitals21. Because of the dynamic nature and recent shift in the etiology and antimicrobial susceptibility pattern of UTIs, it has become essential for the clinicians to keep abreast of the etiological agents and their antimicrobial susceptibility profiles, so as to direct and improvise the preliminary empirical treatment22. In the light of these observations, this study was conducted to evaluate etiology of UTIs in a population of Southern Punjab, Pakistan and antibiotic susceptibility profiling of MDR P. aeruginosa was evaluated. It was observed that UTI was more prevalent in females as compared to males, is due to the differences in the anatomy of their urogenital organs; as short urethra in females allows microbes to gain relatively easy access to bladder so as to cause infection23. In addition, females are inclined towards several circumstances like vaginitis as well as decay and prolapse of vagina or womb which leads towards the development of UTI24. The symptoms of UTI were also more prevalent among females which reinforces the previous observations in this regard25. It has been reported that UTI is the most significant cause of hospital-acquired infections. Among patients who are hospitalized, urinary catheter has been revealed as the major predisposing factor for UTIs26. Accordingly, the most Prevalence and Antibiotic Susceptibility Profiling of MDR P. aeruginosa Vol. 13 (1), June 2022 ISSN (Print): 2305 – 8722 ISSN (Online): 2521 – 8573 R A D S J . B i o l . R e s . A p p l . S c i . 7 substantial risk factor for UTI detected in the present study was presence of indwelling catheter. Previously, this has been reported that the duration of catheterization is directly proportional to the risk of developing UTI27. Another important risk factor observed here was pregnancy which has been commonly reported as a factor responsible for making females vulnerable to the development of UTIs28. We found that majority of the patients were 20 to 30 years old which has been scarcely reported previously and mostly, old age has been documented as a risk factor for UTIs29. This could possibly be explained based on the sexual behavior and use of contraceptive agents30. Conceivably due to the rise in rate of sexual action, as well as associated rise in the number of pregnancies and utilization of various kinds of contraceptive agents, younger individuals were the most affected ones31. However, study on a larger sample population is required to confirm this speculation. E. coli was the most commonly isolated uropathogen in individuals belonging to both genders, and P. aeruginosa was responsible for 7% of the overall cases, approximately. Similar observations have been reported previously in another population of Pakistan32. A vast majority of the P. aeruginosa isolates were revealed as being MDR here which is in line with the emerging data regarding prevalence of MDR uropathogens33. Cefixime was reported here as being the most effective of all the drugs tested against P. aeruginosa whereas amikacin was the least effective owing to high degree of resistance against it. Previously, in various populations the drug resistance patterns have been similar as well as dissimilar implying that antibiotic resistance profile of uropathogens varies across the globe34,35. Over-enthusiastic use of antibiotics has led to the global emergence of resistance among various bacteria represented by recommendation of antibiotics without susceptibility reports or prolonged usage of drugs36. Antibiotics resistance has been declared as global human health issue by WHO and over-ambitious usage of broad-spectrum antibiotics has elevated the rate of drug resistance across the world37. C O N C L U S I O N UTIs account for a considerable burden of antibiotic prescriptions and gradual rise in resistance against commonly used antibiotics needs to be deliberated immediately. Such growing resistance in UTI patients by P. aeruginosa can be addressed by adopting essential management options by physicians as well as medical professionals based on the knowledge of etiology and antibiotic susceptibility of pathogens. Quest for alternative treatment options which may be combined with antimicrobial drugs is the need of the hour. E T H I C A L A P R O V A L Ethical approval for the study was obtained from Ethical Review Board of Nishtar Hospital, Multan, Pakistan. C O N F L I C T O F I N T E R E S T The authors declare no conflict of interest. F U N D I N G S O U R C E The study received no external funding. A C K N O W L E D G E M E N T S We acknowledge the cooperation of study participants who facilitated our work by providing their personal information and samples. 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