Introduction

P. aeruginosa is a gram-negative, encapsulated, motile, rod shaped, and facultative aerobe of the family Pseudomonadaceae [1]. It is found in moist environments, such as water, soil, and skin. However, studies have also reported its continued presence on dry inanimate surfaces for several days or even months [2]. It grows well at 37°C but can survive up to 42°C [3]. It forms characteristically smooth, blue-green colonies with grapes like smell which helps in its identification on the growth media [4, 5]. Its isolates may produce three types of colonies. 'Natural isolates' taken from soil and water typically produce small and rough colonies. Whereas, 'clinical samples' either yield colonies with a fried egg like appearance (large, smooth with flat edges and elevated appearance) or colonies with mucoid appearance (isolates from respiratory and urinary tract secretions) [4, 6]. The two different solubles of P. aeruginosa, namely blue pyocyanin and pyoverdine, are causes of blue pus which is the typical feature of P. aeruginosa suppurative infections which can interfere with the human respiratory epithelium and nasal cilia, thereby leading to pro-inflammatory responses [7].

P. aeruginosa capitalizes on the weakened immune system of human beings and thrives in various vulnerable conditions, such as burn wounds, long-lasting wounds, chronic obstructive pulmonary disorder (COPD), the presence of implanted biomaterials [8], ventilator-associated pneumonia (VAP), bloodstream infections leading to sepsis, infections in soft tissues of burns, open wounds, and post-surgery patients, urinary tract infections (UTIs), diabetic foot ulcers, and keratitis related to the extended use of contact lenses [9]. The primary cause of life-threatening and long-lasting infections in individuals with cystic fibrosis and other lung conditions with high rates of illness and death is primarily attributed to it [10]. When a catheter is inserted, it puts the patient at a higher risk of acquiring a UTI known as catheter-associated urinary tract infection (CAUTI). This is because pathogens utilize the catheter as a platform for colonization and the formation of biofilms [8, 9], which results in bacteriuria and increases the chances of developing secondary bloodstream infections [11]. Among all the healthcare-associated infections, P. aeruginosa has an estimated prevalence of 7.1–7.3% [12–14]; however, different prevalence studies have shown that over the past decade, this rate has been increasing. Moreover, in ICU (intensive care unit) patients, P. aeruginosa accounts for 23% of all the acquired infections [12, 15].

The genome of P. aeruginosa encodes a range of resistant genes, such as multiple MDR efflux pumps and antibiotic inactivating enzymes [16, 17], broad spectrum beta-lactamases that provides resistance against beta-lactam antibiotics, aminoglycosides, fluoroquinolones, carbapenems, chloramphenicol, tetracycline, macrolides, trimethoprim sulfamethoxazole, rifampin, and cephalosporins, making the available antibiotics for its treatment ineffective [17, 18]. Once it enters inside the host it can become challenging to treat because a variety of mechanisms for establishment, adaptation, and survival exist in P. aeruginosa, such as quorum sensing (QS), motility-sessility switch, biofilm formation, antibiotic resistance mechanisms, adaptive radiation for persistence, and the CRISPR-Cas systems [19]. Therefore, its infections can be a serious threat to the healthcare system, worldwide.

The primary methods employed by P. aeruginosa to defend against antibiotic assault can be categorized as intrinsic resistance, acquired resistance, and adaptive resistance [20, 21]. This bacterium possesses inherent mechanisms of resistance, such as reduced permeability of its outer membrane, the presence of efflux pumps that actively expel antibiotics from the cell, and the production of enzymes that can deactivate antibiotics. On the other hand, acquired resistance in P. aeruginosa can occur through the horizontal transfer of resistance genes or through mutational changes in its genetic material. Additionally, this bacterium exhibits adaptive resistance by forming biofilms in the lungs of infected individuals. These biofilms act as barriers, impeding the diffusion of antibiotics and limiting their effectiveness against bacterial cells [21]. Consequently, there is an urgent need to develop new antibiotics or to explore alternative therapeutic strategies to effectively treat P. aeruginosa infections that are resistant to traditional antibiotics [20, 22]. Furthermore, proper preventive measures including screening procedures, patient care and cleaning, proper antibiotic use and waste disposal, hand hygiene, cleaning, isolation, and proper examination and inspection of patients' rooms and other shared facilities such as hydrotherapy suite and physiotherapy room can help control the infections to a large extent ².

2. METHODOLOGY

2.1. Study Design

The current study was a descriptive cross-sectional study conducted at the pathology department of Rehman Medical Institute (RMI), Peshawar. A total of 175 isolates of P. aeruginosa were included in this study using the convenient sampling technique.

2.2. Data Collection Procedure

Samples were collected after obtaining approval from the hospital's ethical and research committees. Individuals of all age groups infected with Pseudomonas who had submitted their samples to the Microbiology Department of RMI were included in the study.

2.3. Sample Collection and Handling

Samples were isolated from six (06) different sites including pus/swab samples, sputum, urine, fluid, blood, and others using proper aseptic techniques, as done by [23, 24]. All samples were properly labeled and were sent to the laboratory for further processing.

2.4. Sample Processing

All the collected samples were inoculated separately on different growth media including blood agar, chocolate agar, MacConkey agar, and CLED (Cysteine-Lactose-Electrolyte-Deficient) agar for their morphological identification. Once inoculated, the samples were incubated at 37°C for 24 hours. Inoculation was followed by the biochemical identification of clinical isolates which was done using Gram staining, catalase, oxidase, SIM (sulfur indole motility), citrate utilization test, and urease test. Later, bacterial inoculums were prepared in comparison with 0.5% of McFarland standard. Furthermore, antibiotic susceptibility testing (AST) was performed on Muller-Hinton agar using the Kirby-Bauer (KB) disc diffusion method.

3. RESULTS

The clinical isolates formed different types of colonies (specific to P. aeruginosa) on different media after incubation at 37°C for 24 hours, which are listed in the table below.

Table 1.Colony Morphology of P. aeruginosa on Different Media

Among 175 samples, the most frequent category was of 'pus/swab' including 69 samples (39.4%), followed by 'others' including 32 samples (18.3%), 'blood' including 29 samples (16.6%), 'urine' including 23 samples (13.1%), and 'sputum' including 16 samples (9.1%). The lowest number of samples were from the 'fluids' category which included only 6 samples (3.4%). This categorization of clinical isolates is indicated in Figure 1 below.

Figure 1.Percentages of Clinical Isolates

Gender-wise distribution of the study population involved a total of 101 male patients and 74 female patients. These male and female patients belonged to different age groups summarized in Table 2.

Antibiotic susceptibility testing (AST) showed that all the P. aeruginosa isolates were sensitive to colistin sulfate (100%). Whereas, 64.6% isolates were sensitive to imipenem, 61.14% to amikacin, 60.6% to meropenem, and 22.5% to levofloxacin. Similarly, by analyzing the resistance pattern of the isolates, it was found that 77.5% were resistant to levofloxacin, 64.1% to ciprofloxacin, 60.4% to gentamicin, 56.7% to aztreonam, 52.6% to cefepime, and 51.6% to ceftazidime. The sensitivity and resistance patterns of these antibiotics are depictd  in Table 3 below.

Table 2. Age-wise Distribution of the Study Population

Table 3. Different Antibiotics Used for AST of P. aeruginosa

4. DISCUSSION

The inherent resistance of P. aeruginosa to many antibiotics is the cause of higher morbidity and mortality rates. This resistance is attributed to the presence of multi-drug efflux systems, production of β-lactamases, as well as reduced outer membrane permeability [25]. Irrespective of the advancements in the sanitation facilities and availability of several antimicrobial agents with anti-pseudomonal potential, life-threating, hospital associated, pseudomonal infections still account for high mortality rates. The antibiotics resistance issue has been greatly amplified over the past few years. Therefore, regular assessment is required to have a clear opinion of the clinical outcome of different therapeutic options [26].

The current study showed a high rate of pseudomonal infection in men as compared to women and this is in line with the studies previously conducted by Abdul Samad in Karachi, Pakistan [27]. In the current study, the age group 41-60 years had the highest frequency of patients. Similar results were also been obtained previously during the studies conducted in AFIP, Rawalpindi [28], where the highest frequency age group was 41-60 years. The AST carried out in the current study indicated 100% susceptibility to colistin sulphate followed by imipenem, while the highest resistance was observed to levofloxacin and ciprofloxacin. This is in accordance with the study conducted in India [29] (except for colistin sulphate used nowhere in the protocol but RMI).

4.1. Conclusion

The study concluded that aminoglycosides showed variable outcomes since the isolates showed resistance to gentamicin and were sensitive to amikacin. Fluoroquinolones, such as levofloxacin and ciprofloxacin, were observed as resistant antibiotics in the current investigation. Whereas, the combinations of antibiotics including cefoperazone-sulbactam and piperacillin-tazobactam showed less effectiveness, as compared to the antibiotics imipenem and meropenem.

Conflict of Interest

The author of the manuscript has no financial or non-financial conflict of interest in the subject matter or materials discussed in this manuscript.

Data Availability Statement

The data associated with this study will be provided by the corresponding author upon request.

FUNDING SOURCE

The authors received no funding for this study.