Antimicrobial Susceptibility Pattern of Pseudomonas Aeruginosa Isolated From Clinical and Environmental Sources In Punjab, Pakistan

The increasing incidence of antimicrobial resistance is a public health concern, and Pseudomonas aeruginosa is known to be resistant to a variety of antibiotics. Objective: Antimicrobial resistance and the multiple antibiotic resistance (MAR) index of P. aeruginosa from environmental and clinical sources were studied in the current study. Method: A total of 170 samples were evaluated, with 85 samples each from environmental sources and clinical settings. The isolates were subjected to microbial analysis and antimicrobial sensitivity testing. The ndings revealed that 45.88 % (39) of the 85 clinical isolates tested for the presence of P. aeruginosa were positive. In terms of prevalence, there were signicant variations (p 0.05) between the clinical samples. Results: Wound samples had the highest isolation rate of 28.2%, while urine samples had the lowest (12.8%). P. aeruginosa was found in 38.8 % (33/85) of the samples isolated from environmental sources. In terms of prevalence, there was a highly signicant difference (p 0.01) between the isolates. All of the positive isolates were completely resistant to cefuroxime and amoxicillin (100 %). The majority were also resistant to, cotrimoxazole (82%), nalidixic acid (82%), ciprooxacin (86%), and tobramycin (69%). There was a substantial variation in the resistance patterns of isolates. Conclusion: The current study demands comprehensive measures to combat antimicrobial resistance in P. aeruginosa.

and has consequently colonized a wide range of natural and arti cial reservoirs. It feeds on a variety of organic materials; in mammals, its adaptability allows it to infect injured tissues or those with weakened immune systems [3]. The bacterium is considered as a signi cant source of infection in a variety of vulnerable persons in hospital settings, including those undergoing critical care (ICU), with burns or neutropenia, bacteremia, wound infections, as well as other cutaneous and systemic infections. Incidence and mortality rates with P. aeruginosa infection may be on the rise among these groups of people. Because P. aeruginosa is resistant to a wide range of antimicrobials, A total of 170 specimens were evaluated, with 85 coming from each clinical and environmental source. Two government-owned hospitals and two privately-owned hospitals in Pakistan's Punjab region provided the samples. The clinical samples included 17 urine samples, 17 sputum samples, 17 wound swab samples, 17 blood samples, and 17 eye swab samples. They came from both hospitalized and non-hospitalized people who came to the clinics. For the collection of clinical samples, ethical approval was obtained. Hospital sinks (17), garbage sites (17), and storage rooms (17) were used to collect environmental samples. All samples were taken in an aseptic manner. Isolation of P. aeruginosa: A 1.0 ml aliquot of the samples was prepared by diluting to the order of 106 following standard serial dilution techniques. Following that, 100 µl of each diluent (102-106) was inoculated on cetrimide agar and incubated for 18-24 hours at 37oC. The puri ed green colonies on cetrimide agar were sub-cultured and cultivated at 37oC for 18-24

M E T H O D S
hours. Following that, colonies were puri ed on nutrient agar and cultured at 37oC for 24-48 hours before being kept on agar slants at 4 oC until ready for analysis [8]. P. aeruginosa isolates were identi ed using VITEK®-MS [9]. Antibiotic Susceptibility Testing is a method of determining whether or not a person is susceptible to different classes of antibiotics. The disc diffusion (Kirby-Bauer) method was used to evaluate antibiotic susceptibility, as indicated by the Clinical and Laboratory Standards Institute's standards (CLSI, 2019). To generate a lawn of bacteria, a single inoculum of each bacterial isolate was emulsi ed in 5.0 mL sterile normal saline in B ou bottles. Mueller-Hinton agar plates were inoculated with sterile cotton swabs soaked in a standardized solution of bacterial cultures. Following that, antibiotic discs (Mast Diagnostics, Merseyside, United Kingdom) containing the following antibiotics were used: amikacin (30 g), gentamicin (10 g), streptomycin (10 g), tobramycin (10 g), ceftazidime (30 g), ceftriaxone (30 g), cefuroxime (30 g), amoxicillin (10 g), imipenem (10 g), and meropenem (10g). To avoid overlapping inhibition zones, discs were spaced at least 15 mm apart and away from the plate borders. The plates were incubated for 18-24 hours at 37°C, following which the zones of inhibition (millimeters) were measured to see if the pro le was sensitive (S), intermediate (I), or resistance (I). The zones were interpreted according to Clinical and Laboratory Standards (CLSI 2019) [10]. SPSS, version 26.0, was used to tabulate and analyze all of the data. The percentages of qualitative variables were used to compare them using charts and the chi-square test. Statistical signi cance was de ned as a p-value of less than 0.05.

R E S U L T S
treating these infections might be problematic. Furthermore, the advent and dissemination of resistant organisms of the bacterium have made treatment regimens challenging [4]. Several P. aeruginosa epidemics in intensive care units (ICUs) have been documented in the literature, indicating the potential for Pseudomonas spp. to develop microbial reservoirs in the healthcare setting. In contrast to other important environmental infections, P. aeruginosa thrives in a wide range of temperatures and has an incredible ability to manipulate and thrive in nutrientpoor environments. It sticks to surfaces thanks to its capsular polysaccharide. When favorable environmental circumstances exist, cells rapidly enter the exponential phase on surfaces connected to water conveyance systems, forming bio lms [5]. The capacity of some P. aeruginosa strains to produce carbapenemases is a major threat. As a result of these di culties, it seems fair to identify the bacterium's sources and reservoirs to avoid its acquisition by both hospitalized and non-hospitalized people [6]. Environmental factors have been responsible for many illnesses linked to P. aeruginosa outbreaks. According to emerging evidence from possible investigations, environmental factors may, however, have a role in the epidemiology of occasional P. aeruginosa infections in clinical settings [7]. Antimicrobial resistance analysis of clinical and environmental reservoirs in pseudomonas isolates will allow the possibilities of new methods and re ning of current ones to stop transmission from these sources. As a result, the goal of this investigation was to determine P. aeruginosa antibiogram pro ling from clinical and environmental sources.  . P. aeruginosa has many characteristics that help it survive in a wide variety of environments. Through the mechanism of multidrug e ux pumps, the organism is naturally resistant to a variety of disinfectants, including biguanides and quaternary ammonium compounds. In addition, the organism's capacity to develop bio lm on inanimate surfaces favors disinfectant resistance while also preventing manual removal. Its ability to survive in damp conditions is aided by the type III secretion system, which kills free-living amoeba that feeds on environmental bacteria [12]. In this study, a high %age of clinical and environmental isolates was resistant to the majority of antibiotics, including penicillin, cephalosporins, ceftriaxone, and cefuroxime (20% -100 %), which is consistent with other studies. The formation of betalactamase enzymes, which break down the beta-lactam ring, may cause high resistance to beta-lactam antibiotics in those isolates. Pseudomonas species are naturally resistant to penicillins, cephems, and rifampin because they contain a moderately impermeable membrane, inducible e ux mechanisms, and a chromosomally encoded inducible beta-lactamase.
[13]. In both clinical and environmental isolates, amoxicillin and cefuroxime had the highest antibiotic resistance, whereas imipenem, meropenem, amikacin, and ceftazidime had the lowest. Clinical and environmental isolates of P. aeruginosa were s h o w n t o b e 1 0 0 % r e s i s t a n t t o c e fo t a x i m e , chloramphenicol, penicillin, ampicillin, doxycycline, erythromycin, tetracycline, and cloxacillin, according to Haleem et al. (2011) [14]. Resistance to imipenem was found in 3% of clinical isolates and 21% of environmental isolates.   Table 3 shows the antibiotic susceptibility pro les of clinical and environmental isolates. All of the positive clinical isolates were completely resistant to cefuroxime and amoxicillin (100 %). The majority of the clinical isolates were also resistant to nalidixic acid (82%), cotrimoxazole (82%), and cipro oxacin (82%). A p-value of 0.001 is indicating there was a signi cant variation in resistance patterns across clinical isolates. Imipenem (94 %), meropenem (77 %), ceftazidime (77 %), and amikacin (74 %) were the most sensitive antibiotics among the clinical isolates (Table 3). Cefuroxime (100%), amoxicillin (100%), tetracycline (100%), and chloramphenicol (94%) resistance were found in all environmental isolates. At p0.001, the resistance patterns of the environmental isolates showed a very high signi cant difference. However, ceftazidime (79%) amikacin (70%), and imipenem (64%) were all effective against some of the environmental isolates (Table  3).   The limited usage of imipenem has resulted in a decrease in resistance to this antibiotic. The ndings of this investigation revealed a signi cant rise in quinolone resistance (cipro oxacin, o oxacin, and nalidixic acid). For clinical and environmental isolates, nalidixic acid demonstrated the highest resistance (82 % and 85 %), followed by cipro oxacin (80 % and 79 %) and o oxacin (67 % and 55 %). This means that in this setting, quinolones alone cannot be relied upon as an antipseudomonal antibiotic. Because of the rising resistance of nalidixic acid in many hospitals, its empirical use is either prohibited or restricted to reduce the rising resistance rates [15]. It's worth noting that all the environmental isolates were tetracycline resistant. Tetracycline's amazing multiresistances might be due to the antibiotic's widespread abuse in our environment, as well as an innate and acquired resistance mechanism generated primarily by an active e ux system, which e ciently expels the molecule from the cell. The resistance among P. aeruginosa to a variety of antibiotics might be the consequence of gene transfer into the hospital environment, which is a prevalent nosocomial occurrence, as well as antibiotic overuse. There was no signi cant difference in resistance patterns of clinical and environmental isolates to the majority of antibiotics (P>0.05) in this investigation. Multiple antibiotic resistances (MARs) were found in all of the isolates evaluated in this investigation, ranging from four to sixteen antibiotics spread over three to seven classes. A previous study reported a MAR of ve to eleven antibiotics in their trials. MAR bacterial strains can also evolve as a consequence of independent processes accumulating sequentially in an organism [16][17][18][19][20]. The nding suggests that the isolates in this investigation came from a high-risk source(s) of contamination. Multidrug resistance in environmental isolates might be connected to the uncontrolled release of antibiotics and toxins into the environment, putting selective pressure on these medications. Antibiotic usage in hospitals and the general public exert strong selection pressure on antibioticresistant microorganisms.

D I S C U S S I O N
The nding of the current study revealed that P. aeruginosa is very widespread, and may be isolated from a variety of clinical and environmental sources in different biological habitats. The isolates were resistant to the majority of antip s e u d o m o n a l m e d i c a t i o n s e x a m i n e d , a n d t h e contamination came from high-risk sources. The wide range of resistance phenotypes found in clinical and environmental samples has led to assumptions regarding inherent gene transfer in natural microbial ecosystems.